Angiopoietin-like 3 (ANGPTL3) iRNA compositions and methods of use thereof

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting the ANGPTL3 gene, as well as methods of inhibiting expression of ANGPTL3 and methods of treating subjects having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia, using such dsRNA compositions.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No. 15/068,912, filed on Mar. 14, 2016, which is a continuation of U.S. patent application Ser. No. 14/132,999, now U.S. Pat. No. 9,322,018, issued on Apr. 26, 2016, which is a 35 U.S.C. 111(a) continuation application, which claims priority to PCT/US2012/043378, filed on Jun. 20, 2012, U.S. Provisional Application No. 61/499,620, filed on Jun. 21, 2011, and to U.S. Provisional Application No. 61/638,288, filed on Apr. 25, 2012. The entire contents of each of the foregoing are hereby incorporated herein by reference.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Jul. 26, 2017, is named Seq_Listing_121301_00304.txt and is 444,428 bytes in size.

BACKGROUND OF THE INVENTION

Angiopoietin-like 3 (ANGPTL3) is a member of the angiopoietin-like family of secreted factors that regulates lipid metabolism and that is predominantly expressed in the liver (Koishi, R. et al., (2002) Nat. Genet. 30(2):151-157). ANGPTL3 dually inhibits the catalytic activities of lipoprotein lipase (LPL), which catalyzes the hydrolysis of triglycerides, and of endothelial lipase (EL), which hydrolyzes high density lipoprotein (HDL) phospholipids. In hypolipidemic, yet obese, KK/Snk mice, a reduction in ANGPTL3 expression has a protective effect against hyperlipidemia and artherosclerosis by promoting the clearance of triglycerides (Ando et al., (2003) J. Lipid Res., 44:1216-1223). Human ANGPTL3 plasma concentrations positively correlate with plasma HDL cholesterol and HDL phospholipid levels (Shimamura et al., (2007) Arterioscler. Thromb. Vasc. Biol., 27:366-372).

Disorders of lipid metabolism can lead to elevated levels of serum lipids, such as triglycerides and/or cholesterol. Elevated serum lipids are strongly associated with high blood pressure, cardiovascular disease, diabetes and other pathologic conditions. Hypertriglyceridemia is an example of a lipid metabolism disorder that is characterized by high blood levels of triglycerides. It has been associated with atherosclerosis, even in the absence of high cholesterol levels (hypercholesterolemia). When triglyceride concentrations are excessive (i.e., greater than 1000 mg/dl or 12 mmol/1), hypertriglyceridemia can also lead to pancreatitis. Hyperlipidemia is another example of a lipid metabolism disorder that is characterized by elevated levels of any one or all lipids and/or lipoproteins in the blood. Current treatments for disorders of lipid metabolism, including dieting, exercise and treatment with statins and other drugs, are not always effective. Accordingly, there is a need in the art for alternative treatments for subjects having disorders of lipid metabolism.

SUMMARY OF THE INVENTION

The present invention provides iRNA compositions which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPL3 gene. The ANGPL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPL3 gene and/or for treating a subject who would benefit from inhibiting or reducing the expression of an ANGPL3 gene, e.g., a subject suffering or prone to suffering from a disorder of lipid metabolism, such as a subject suffering or prone to suffering from hyperlipidemia or hypertriglyceridemia.

Accordingly, in one aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, wherein the sense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:1 and the antisense strand comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the nucleotide sequence of SEQ ID NO:5.

In another aspect, the present invention provides double-stranded ribonucleic acids (dsRNAs) for inhibiting expression of ANGPTL3. The dsRNAs comprise a sense strand and an antisense strand, the antisense strand comprising a region of complementarity which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from any one of the antisense sequences listed in Tables 2, 3, 7, 8, 9 and 10.

In one embodiment, the sense and antisense strands comprise sequences selected from the group consisting of AD-53063.1, AD-53001.1, AD-53015.1, AD-52986.1, AD-52981.1, AD-52953.1, AD-53024.1, AD-53033.1, AD-53030.1, AD-53080.1, AD-53073.1, AD-53132.1, AD-52983.1, AD-52954.1, AD-52961.1, AD-52994.1, AD-52970.1, AD-53075.1, AD-53147.1, AD-53077.1 of Tables 7 and 8.

In certain embodiments of the invention, the dsRNAs comprise at least one modified nucleotide. In one embodiment, at least one of the modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group. In another embodiment, the modified nucleotide is selected from the group consisting of a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modified nucleotide, a locked nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modified nucleotide, a morpholino nucleotide, a phosphoramidate, and a non-natural base comprising nucleotide.

The region of complementarity of the dsRNAs may be at least 17 nucleotides in length, between 19 and 21 nucleotides in length, or 19 nucleotides in length.

In one embodiment, each strand of a dsRNA is no more than 30 nucleotides in length.

At least one strand of a dsRNA may comprise a 3′ overhang of at least 1 nucleotide or at least 2 nucleotides.

In certain embodiments, a dsRNA further comprises a ligand. In one embodiment, the ligand is conjugated to the 3′ end of the sense strand of the dsRNA.

In some embodiments, the ligand is one or more N-acetylgalactosamine (GalNAc) derivatives attached through a bivalent or trivalent branched linker. In particular embodiments, the ligand is

In some embodiments, the RNAi agent is conjugated to the ligand as shown in the following schematic

In some embodiments, the RNAi agent further includes at least one phosphorothioate or methylphosphonate internucleotide linkage. In some embodiments, the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminal of one strand. In some embodiments, the strand is the antisense strand. In other embodiments, the strand is the sense strand.

In one embodiment, the region of complementarity of a dsRNA consists of one of the antisense sequences of Tables 2, 3, 7, 8, 9 and 10.

In another embodiment, a dsRNA comprises a sense strand consisting of a sense strand sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10, and an antisense strand consisting of an antisense sequence selected from the sequences of Tables 2, 3, 7, 8, 9 and 10.

In another aspect, the present invention provides a cell, e.g., a hepatocyte, containing a dsRNA of the invention.

In yet another aspect, the present invention provides a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage. The region of complementarity may be least 15 nucleotides in length or 19 to 21 nucleotides in length.

In a further aspect, the present invention provides a cell comprising a vector encoding at least one strand of a dsRNA, wherein the dsRNA comprises a region of complementarity to at least a part of an mRNA encoding ANGPTL3, wherein the dsRNA is 30 base pairs or less in length, and wherein the dsRNA targets the mRNA for cleavage.

In one aspect, the present invention provides a pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising a dsRNA or vector of the invention.

In one embodiment, the pharmaceutical composition comprises a lipid formulation, such as a MC3, SNALP or XTC formulation.

In another aspect, the present invention provides methods of inhibiting ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA or a vector of the invention, and maintaining the cell produced for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.

The cell may be within a subject, such as a human subject, for example a human subject suffering from a disorder of lipid metabolism, e.g., hyperlipidemia or hypertriglyceridemia.

In one embodiment of the methods of the invention, ANGPTL3 expression is inhibited by at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

In another aspect, the present invention provides methods of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby treating the subject.

The disorder may be disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia

In one embodiment, the administration of the dsRNA to the subject causes a decrease in the level of a serum lipid, triglycerides, cholesterol and/or free fatty acids; and/or a decrease in ANGPTL3 protein accumulation. In one embodiment, administration of the dsRNA to the subject causes a decrease in the level of LDL-C, HDL-C, VLDL-C, IDL-C and/or total cholesterol.

In one embodiment, the dsRNA is administered at a dose of about 0.01 mg/kg to about 10 mg/kg, e.g., about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In another aspect, the present invention provides methods of inhibiting the expression of ANGPTL3 in a subject. The methods include administering to the subject a therapeutically effective amount of a dsRNA or a vector of the invention, thereby inhibiting the expression of ANGPTL3 in the subject.

In yet another aspect, the invention provides kits for performing the methods of the invention. In one aspect, the invention provides a kit for performing a method of inhibiting expression of ANGPTL3 gene in a cell by contacting a cell with a double stranded RNAi agent in an amount effective to inhibit expression of the ANGPTL3 in the cell. The kit comprises an RNAi agent and instructions for use and, optionally, means for administering the RNAi agent to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic of the experimental procedure used for in vivo tests described in Example 2.

FIG. 2A is a graph showing measured levels of ANGPTL3 protein in WT mice after treatment with the indicated iRNA or a control.

FIG. 2B is a graph showing measured levels of ANGPTL3 protein in ob/ob mice after treatment with the indicated iRNA or a control.

FIG. 3A is a graph showing measured levels of LDL-c in WT mice after treatment with the indicated iRNA or a control.

FIG. 3B is a graph showing measured levels of LDL-c in ob/ob mice after treatment with the indicated iRNA or a control.

FIG. 4A is a graph showing measured levels of triglycerides in WT mice after treatment with the indicated iRNA or a control.

FIG. 4B is a graph showing measured levels of triglycerides in ob/ob mice after treatment with the indicated iRNA or a control.

FIG. 5A is a graph showing measured levels of total cholesterol (TC) in WT mice after treatment with the indicated iRNA or a control.

FIG. 5B is a graph showing measured levels of total cholesterol (TC) in ob/ob mice after treatment with the indicated iRNA or a control.

FIG. 6A is a graph showing measured levels of HDL-c in WT mice after treatment with the indicated iRNA or a control.

FIG. 6B is a graph showing measured levels of HDL-c in ob/ob mice after treatment with the indicated iRNA or a control.

FIG. 7 is a graph showing measured levels of ANGPTL3 protein in human PCS transgenic mice after treatment with a single dose of the indicated iRNA or a control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides iRNA compositions, which effect the RNA-induced silencing complex (RISC)-mediated cleavage of RNA transcripts of an ANGPTL3gene. The ANGPTL3 gene may be within a cell, e.g., a cell within a subject, such as a human. The present invention also provides methods of using the iRNA compositions of the invention for inhibiting the expression of an ANGPTL3gene and/or for treating a subject having a disorder that would benefit from inhibiting or reducing the expression of an ANGPTL3gene, e.g., a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.

The iRNAs of the invention include an RNA strand (the antisense strand) having a region which is about 30 nucleotides or less in length, e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, which region is substantially complementary to at least part of an mRNA transcript of an ANGPTL3 gene. The use of these iRNAs enables the targeted degradation of mRNAs of an ANGPTL3 gene in mammals. Very low dosages of ANGPTL3 iRNAs, in particular, can specifically and efficiently mediate RNA interference (RNAi), resulting in significant inhibition of expression of an ANGPTL3 gene. Using cell-based assays, the present inventors have demonstrated that iRNAs targeting ANGPTL3 can mediate RNAi, resulting in significant inhibition of expression of an ANGPTL3 gene. Thus, methods and compositions including these iRNAs are useful for treating a subject who would benefit by a reduction in the levels and/or activity of an ANGPTL3 protein, such as a subject having a disorder of lipid metabolism, such as hyperlipidemia or hypertriglyceridemia.

The following detailed description discloses how to make and use compositions containing iRNAs to inhibit the expression of an ANGPTL3 gene, as well as compositions and methods for treating subjects having diseases and disorders that would benefit from inhibition and/or reduction of the expression of this gene.

I. Definitions

In order that the present invention may be more readily understood, certain terms are first defined. In addition, it should be noted that whenever a value or range of values of a parameter are recited, it is intended that values and ranges intermediate to the recited values are also intended to be part of this invention.

The articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeably with, the phrase “including but not limited to”. The term “or” is used herein to mean, and is used interchangeably with, the term “and/or,” unless context clearly indicates otherwise.

The term “ANGPTL3” refers to an angiopoietin like protein 3 having an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless specified otherwise. The term also refers to fragments and variants of native ANGPTL3 that maintain at least one in vivo or in vitro activity of a native ANGPTL3. The term encompasses full-length unprocessed precursor forms of ANGPTL3 as well as mature forms resulting from post-translational cleavage of the signal peptide and forms resulting from proteolytic processing of the fibrinogen-like domain. The sequence of a human ANGPTL3 mRNA transcript can be found at, for example, GenBank Accession No. GI: 41327750 (NM_014495.2; SEQ ID NO:1). The predicted sequence of rhesus ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 297278846 (XM_001086114.2; SEQ ID NO:2). The sequence of mouse ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 142388354 (NM_013913.3; SEQ ID NO:3). The sequence of rat ANGPTL3 mRNA can be found at, for example, GenBank Accession No. GI: 68163568 (NM_001025065.1; SEQ ID NO:4).

The term“ANGPTL3” as used herein also refers to a particular polypeptide expressed in a cell by naturally occurring DNA sequence variations of the ANGPTL3 gene, such as a single nucleotide polymorphism in the ANGPTL3 gene. Numerous SNPs within the ANGPTL3 gene have been identified and may be found at, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp). Non-limiting examples of SNPs within the ANGPTL3 gene may be found at, NCBI dbSNP Accession Nos. rs193064039; rs192778191; rs192764027; rs192528948; rs191931953; rs191293319; rs191171206; rs191145608; rs191086880; rs191012841; or rs190255403.

As used herein, “target sequence” refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3gene, including mRNA that is a product of RNA processing of a primary transcription product. In one embodiment, the target portion of the sequence will be at least long enough to serve as a substrate for iRNA-directed cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during the transcription of an ANGPTL3gene.

The target sequence may be from about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length. For example, the target sequence can be from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

As used herein, the term “strand comprising a sequence” refers to an oligonucleotide comprising a chain of nucleotides that is described by the sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide that contains guanine, cytosine, adenine, thymidine and uracil as a base, respectively. However, it will be understood that the term “ribonucleotide” or “nucleotide” can also refer to a modified nucleotide, as further detailed below, or a surrogate replacement moiety. The skilled person is well aware that guanine, cytosine, adenine, and uracil can be replaced by other moieties without substantially altering the base pairing properties of an oligonucleotide comprising a nucleotide bearing such replacement moiety. For example, without limitation, a nucleotide comprising inosine as its base can base pair with nucleotides containing adenine, cytosine, or uracil. Hence, nucleotides containing uracil, guanine, or adenine can be replaced in the nucleotide sequences of dsRNA featured in the invention by a nucleotide containing, for example, inosine. In another example, adenine and cytosine anywhere in the oligonucleotide can be replaced with guanine and uracil, respectively to form G-U Wobble base pairing with the target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

The terms “iRNA”, “RNAi agent,” “iRNA agent,”, “RNA interference agent” as used interchangeably herein, refer to an agent that contains RNA as that term is defined herein, and which mediates the targeted cleavage of an RNA transcript via an RNA-induced silencing complex (RISC) pathway. iRNA directs the sequence-specific degradation of mRNA through a process known as RNA interference (RNAi). The iRNA modulates, e.g., inhibits, the expression of ANGPTL3 in a cell, e.g., a cell within a subject, such as a mammalian subject.

In one embodiment, an RNAi agent of the invention includes a single stranded RNA that interacts with a target RNA sequence, e.g., an ANGPTL3 target mRNA sequence, to direct the cleavage of the target RNA. Without wishing to be bound by theory, long double stranded RNA introduced into cells is broken down into siRNA by a Type III endonuclease known as Dicer (Sharp et al., Genes Dev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into 19-23 base pair short interfering RNAs with characteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs are then incorporated into an RNA-induced silencing complex (RISC) where one or more helicases unwind the siRNA duplex, enabling the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in one aspect the invention relates to a single stranded RNA (siRNA) generated within a cell and which promotes the formation of a RISC complex to effect silencing of the target gene, i.e., an ANGPTL3 gene. Accordingly, the term “siRNA” is also used herein to refer to an RNAi as described above.

In another aspect, the RNAi agent is a single-stranded antisense RNA molecule. An antisense RNA molecule is complementary to a sequence within the target mRNA. Antisense RNA can inhibit translation in a stoichiometric manner by base pairing to the mRNA and physically obstructing the translation machinery, see Dias, N. et al., (2002) Mol. Cancer Ther. 1:347-355. The single-stranded antisense RNA molecule may be about 13 to about 30 nucleotides in length and have a sequence that is complementary to a target sequence. For example, the single-stranded antisense RNA molecule may comprise a sequence that is at least about 13, 14, 15, 16, 17, 18, 19, 20, or more contiguous nucleotides from one of the antisense sequences in Tables 2, 3, 7, 8, 9 and 10.

In another embodiment, an “iRNA” for use in the compositions and methods of the invention is a double-stranded RNA and is referred to herein as a “double stranded RNAi agent,” “double-stranded RNA (dsRNA) molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA”, refers to a complex of ribonucleic acid molecules, having a duplex structure comprising two anti-parallel and substantially complementary nucleic acid strands, referred to as having “sense” and “antisense” orientations with respect to a target RNA, i.e., an ANGPTL3 gene. In some embodiments of the invention, a double-stranded RNA (dsRNA) triggers the degradation of a target RNA, e.g., an mRNA, through a post-transcriptional gene-silencing mechanism referred to herein as RNA interference or RNAi.

The duplex region may be of any length that permits specific degradation of a desired target RNA through a RISC pathway, and may range from about 9 to 36 base pairs in length, e.g., about 15-30 base pairs in length, for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

The two strands forming the duplex structure may be different portions of one larger RNA molecule, or they may be separate RNA molecules. Where the two strands are part of one larger molecule, and therefore are connected by an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting RNA chain is referred to as a “hairpin loop.” A hairpin loop can comprise at least one unpaired nucleotide. In some embodiments, the hairpin loop can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23 or more unpaired nucleotides.

Where the two substantially complementary strands of a dsRNA are comprised by separate RNA molecules, those molecules need not, but can be covalently connected. Where the two strands are connected covalently by means other than an uninterrupted chain of nucleotides between the 3′-end of one strand and the 5′-end of the respective other strand forming the duplex structure, the connecting structure is referred to as a “linker.” The RNA strands may have the same or a different number of nucleotides. The maximum number of base pairs is the number of nucleotides in the shortest strand of the dsRNA minus any overhangs that are present in the duplex. In addition to the duplex structure, an RNAi may comprise one or more nucleotide overhangs.

As used herein, the term “nucleotide overhang” refers to at least one unpaired nucleotide that protrudes from the duplex structure of an iRNA, e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNA extends beyond the 5′-end of the other strand, or vice versa, there is a nucleotide overhang. A dsRNA can comprise an overhang of at least one nucleotide; alternatively the overhang can comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides or more. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. In another embodiment, one or more of the nucleotides in the overhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to a dsRNA mean that there are no unpaired nucleotides or nucleotide analogs at a given terminal end of a dsRNA, i.e., no nucleotide overhang. One or both ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt, the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNA is a dsRNA that is blunt at both ends, i.e., no nucleotide overhang at either end of the molecule. Most often such a molecule will be double-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of an iRNA, e.g., a dsRNA, which includes a region that is substantially complementary to a target sequence, e.g., an ANGPTL3 mRNA. As used herein, the term “region of complementarity” refers to the region on the antisense strand that is substantially complementary to a sequence, for example a target sequence, e.g., an ANGPTL3 nucleotide sequence, as defined herein. Where the region of complementarity is not fully complementary to the target sequence, the mismatches can be in the internal or terminal regions of the molecule. Generally, the most tolerated mismatches are in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5′- and/or 3′-terminus of the iRNA.

The term “sense strand” or “passenger strand” as used herein, refers to the strand of an iRNA that includes a region that is substantially complementary to a region of the antisense strand as that term is defined herein.

As used herein, and unless otherwise indicated, the term “complementary,” when used to describe a first nucleotide sequence in relation to a second nucleotide sequence, refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide sequence to hybridize and form a duplex structure under certain conditions with an oligonucleotide or polynucleotide comprising the second nucleotide sequence, as will be understood by the skilled person. Such conditions can, for example, be stringent conditions, where stringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g., “Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions as can be encountered inside an organism, can apply. The skilled person will be able to determine the set of conditions most appropriate for a test of complementarity of two sequences in accordance with the ultimate application of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA as described herein, include base-pairing of the oligonucleotide or polynucleotide comprising a first nucleotide sequence to an oligonucleotide or polynucleotide comprising a second nucleotide sequence over the entire length of one or both nucleotide sequences. Such sequences can be referred to as “fully complementary” with respect to each other herein. However, where a first sequence is referred to as “substantially complementary” with respect to a second sequence herein, the two sequences can be fully complementary, or they can form one or more, but generally not more than 5, 4, 3 or 2 mismatched base pairs upon hybridization for a duplex up to 30 base pairs, while retaining the ability to hybridize under the conditions most relevant to their ultimate application, e.g., inhibition of gene expression via a RISC pathway. However, where two oligonucleotides are designed to form, upon hybridization, one or more single stranded overhangs, such overhangs shall not be regarded as mismatches with regard to the determination of complementarity. For example, a dsRNA comprising one oligonucleotide 21 nucleotides in length and another oligonucleotide 23 nucleotides in length, wherein the longer oligonucleotide comprises a sequence of 21 nucleotides that is fully complementary to the shorter oligonucleotide, can yet be referred to as “fully complementary” for the purposes described herein.

“Complementary” sequences, as used herein, can also include, or be formed entirely from, non-Watson-Crick base pairs and/or base pairs formed from non-natural and modified nucleotides, in so far as the above requirements with respect to their ability to hybridize are fulfilled. Such non-Watson-Crick base pairs include, but are not limited to, G:U Wobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantially complementary” herein can be used with respect to the base matching between the sense strand and the antisense strand of a dsRNA, or between the antisense strand of an iRNA agent and a target sequence, as will be understood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary to at least part of” a messenger RNA (mRNA) refers to a polynucleotide that is substantially complementary to a contiguous portion of the mRNA of interest (e.g., an mRNA encoding ANGPTL3). For example, a polynucleotide is complementary to at least a part of an ANGPTL3mRNA if the sequence is substantially complementary to a non-interrupted portion of an mRNA encoding ANGPTL3.

In general, the majority of nucleotides of each strand are ribonucleotides, but as described in detail herein, each or both strands can also include one or more non-ribonucleotides, e.g., a deoxyribonucleotide and/or a modified nucleotide. In addition, an “iRNA” may include ribonucleotides with chemical modifications. Such modifications may include all types of modifications disclosed herein or known in the art. Any such modifications, as used in an iRNA molecule, are encompassed by “iRNA” for the purposes of this specification and claims.

The term “inhibiting,” as used herein, is used interchangeably with “reducing,” “silencing,” “downregulating,” “suppressing” and other similar terms, and includes any level of inhibition.

The phrase “inhibiting expression of an ANGPTL3,” as used herein, includes inhibition of expression of any ANGPTL3 gene (such as, e.g., a mouse ANGPTL3 gene, a rat ANGPTL3 gene, a monkey ANGPTL3 gene, or a human ANGPTL3 gene) as well as variants or mutants of an ANGPTL3 gene that encode an ANGPTL3 protein.

“Inhibiting expression of an ANGPTL3 gene” includes any level of inhibition of an ANGPTL3 gene, e.g., at least partial suppression of the expression of an ANGPTL3 gene, such as an inhibition by at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of an ANGPTL3 gene may be assessed based on the level of any variable associated with ANGPTL3 gene expression, e.g., ANGPTL3 mRNA level or ANGPTL3 protein level. The expression of an ANGPTL3 may also be assessed indirectly based on the levels of a serum lipid, a triglyceride, cholesterol (including LDL-C, HDL-C, VLDL-C, IDL-C and total cholesterol), or free fatty acids. Inhibition may be assessed by a decrease in an absolute or relative level of one or more of these variables compared with a control level. The control level may be any type of control level that is utilized in the art, e.g., a pre-dose baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as, e.g., buffer only control or inactive agent control).

In one embodiment, at least partial suppression of the expression of an ANGPTL3 gene, is assessed by a reduction of the amount of ANGPTL3 mRNA which can be isolated from or detected in a first cell or group of cells in which an ANGPTL3 gene is transcribed and which has or have been treated such that the expression of an ANGPTL3 gene is inhibited, as compared to a second cell or group of cells substantially identical to the first cell or group of cells but which has or have not been so treated (control cells). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, as used herein, includes contacting a cell by any possible means. Contacting a cell with an RNAi agent includes contacting a cell in vitro with the iRNA or contacting a cell in vivo with the iRNA. The contacting may be done directly or indirectly. Thus, for example, the RNAi agent may be put into physical contact with the cell by the individual performing the method, or alternatively, the RNAi agent may be put into a situation that will permit or cause it to subsequently come into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating the cell with the RNAi agent. Contacting a cell in vivo may be done, for example, by injecting the RNAi agent into or near the tissue where the cell is located, or by injecting the RNAi agent into another area, e.g., the bloodstream or the subcutaneous space, such that the agent will subsequently reach the tissue where the cell to be contacted is located. For example, the RNAi agent may contain and/or be coupled to a ligand, e.g., GalNAc3, that directs the RNAi agent to a site of interest, e.g., the liver. Combinations of in vitro and in vivo methods of contacting are also possible. For example, a cell may also be contacted in vitro with an RNAi agent and subsequently transplanted into a subject.

In one embodiment, contacting a cell with an iRNA includes “introducing” or “delivering the iRNA into the cell” by facilitating or effecting uptake or absorption into the cell. Absorption or uptake of an iRNA can occur through unaided diffusive or active cellular processes, or by auxiliary agents or devices. Introducing an iRNA into a cell may be in vitro and/or in vivo. For example, for in vivo introduction, iRNA can be injected into a tissue site or administered systemically. In vivo delivery can also be done by a beta-glucan delivery system, such as those described in U.S. Pat. Nos. 5,032,401 and 5,607,677, and U.S. Publication No. 2005/0281781, the entire contents of which are hereby incorporated herein by reference. In vitro introduction into a cell includes methods known in the art such as electroporation and lipofection. Further approaches are described herein below and/or are known in the art.

The term “SNALP” refers to a stable nucleic acid-lipid particle. A SNALP is a vesicle of lipids coating a reduced aqueous interior comprising a nucleic acid such as an iRNA or a plasmid from which an iRNA is transcribed. SNALPs are described, e.g., in U.S. Patent Application Publication Nos. 20060240093, 20070135372, and in International Application No. WO 2009082817, the entire contents of which are hereby incorporated herein by reference. Examples of “SNALP” formulations are described below.

As used herein, a “subject” is an animal, such as a mammal, including a primate (such as a human, a non-human primate, e.g., a monkey, and a chimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, a horse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog, a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or a goose). In an embodiment, the subject is a human, such as a human being treated or assessed for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human at risk for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; a human having a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression; and/or human being treated for a disease, disorder or condition that would benefit from reduction in ANGPTL3 expression as described herein. As used herein, the terms “treating” or “treatment” refer to a beneficial or desired result including, such as lowering levels of triglycerides in a subject. The terms “treating” or “treatment” also include, but are not limited to, alleviation or amelioration of one or more symptoms of a disorder of lipid metabolism, such as, e.g., a decrease in the size of eruptive xanthomas. “Treatment” can also mean prolonging survival as compared to expected survival in the absence of treatment.

By “lower” in the context of a disease marker or symptom is meant a statistically significant decrease in such level. The decrease can be, for example, at least 10%, at least 20%, at least 30%, at least 40% or more, and is preferably down to a level accepted as within the range of normal for an individual without such disorder. As used herein, “prevention” or “preventing,” when used in reference to a disease, disorder or condition thereof, that would benefit from a reduction in expression of an ANGPTL3 gene, refers to a reduction in the likelihood that a subject will develop a symptom associated with such disease, disorder, or condition, e.g., high triglyceride levels or eruptive xanthoma. The likelihood of developing a high tryglyceride levels or eruptive xanthoma is reduced, for example, when an individual having one or more risk factors for a high tryglyceride levels or eruptive xanthoma either fails to develop high tryglyceride levels or eruptive xanthoma or develops high tryglyceride levels or eruptive xanthoma with less severity relative to a population having the same risk factors and not receiving treatment as described herein. The failure to develop a disease, disorder or condition, or the reduction in the development of a symptom associated with such a disease, disorder or condition i (e.g., by at least about 10% on a clinically accepted scale for that disease or disorder), or the exhibition of delayed symptoms delayed (e.g., by days, weeks, months or years) is considered effective prevention.

As used herein, the term “serum lipid” refers to any major lipid present in the blood. Serum lipids may be present in the blood either in free form or as a part of a protein complex, e.g., a lipoprotein complex. Non-limiting examples of serum lipids may include triglycerides and cholesterol, such as total cholesterol (TG), low density lipoprotein cholesterol (LDL-C), high-density lipoprotein cholesterol (HDL-C), very low density lipoprotein cholesterol (VLDL-C) and intermediate-density lipoprotein cholesterol (IDL-C).

As used herein, a “disorder of lipid metabolism” refers to any disorder associated with or caused by a disturbance in lipid metabolism. For example, this term includes any disorder, disease or condition that can lead to hyperlipidemia, or condition characterized by abnormal elevation of levels of any or all lipids and/or lipoproteins in the blood. This term refers to an inherited disorder, such as familial hypertriglyceridemia, or an acquired disorder, such as a disorder acquired as a result of a diet or intake of certain drugs. Exemplary disorders of lipid metabolism include, but are not limited to, atherosclerosis, dyslipidemia, hypertriglyceridemia (including drug-induced hypertriglyceridemia, diuretic-induced hypertriglyceridemia, alcohol-induced hypertriglyceridemia, β-adrenergic blocking agent-induced hypertriglyceridemia, estrogen-induced hypertriglyceridemia, glucocorticoid-induced hypertriglyceridemia, retinoid-induced hypertriglyceridemia, cimetidine-induced hypertriglyceridemia, and familial hypertriglyceridemia), acute pancreatitis associated with hypertriglyceridemia, chylomicron syndrom, familial chylomicronemia, Apo-E deficiency or resistance, LPL deficiency or hypoactivity, hyperlipidemia (including familial combined hyperlipidemia), hypercholesterolemia, gout associated with hypercholesterolemia, xanthomatosis (subcutaneous cholesterol deposits).

Cardiovascular diseases associated with disorders of lipid metabolism are also considered “disorders of lipid metabolism”, as defined herein. These diseases may include coronary artery disease (also called ischemic heart disease), inflammation associated with coronary artery disease, restenosis, peripheral vascular diseases, and stroke.

Disorders related to body weight are also considered “disorders of lipid metabolism”, as defined herein. Such disorders may include obesity, metabolic syndrome including independent components of metabolic syndrome (e.g., central obesity, FBG/pre-diabetes/diabetes, hypercholesterolemia, hypertriglyceridemia, and hypertension), hypothyroidism, uremia, and other conditions associated with weight gain (including rapid weight gain), weight loss, maintenance of weight loss, or risk of weight regain following weight loss.

Blood sugar disorders are further considered “disorders of lipid metabolism”, as defined herein. Such disorders may include diabetes, hypertension, and polycystic ovarian syndrome related to insulin resistance. Other exemplary disorders of lipid metabolism may also include renal transplantation, nephrotic syndrome, Cushing's syndrome, acromegaly, systemic lupus erythematosus, dysglobulinemia, lipodystrophy, glycogenosis type I, and Addison's disease.

“Therapeutically effective amount,” as used herein, is intended to include the amount of an RNAi agent that, when administered to a subject having a disorder of lipid metabolism, is sufficient to effect treatment of the disease (e.g., by diminishing, ameliorating or maintaining the existing disease or one or more symptoms of disease). The “therapeutically effective amount” may vary depending on the RNAi agent, how the agent is administered, the disease and its severity and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended to include the amount of an iRNA that, when administered to a subject having a disorder of lipid metabolism, is sufficient to prevent or ameliorate the disease or one or more symptoms of the disease. Ameliorating the disease includes slowing the course of the disease or reducing the severity of later-developing disease. The “prophylactically effective amount” may vary depending on the iRNA, how the agent is administered, the degree of risk of disease, and the history, age, weight, family history, genetic makeup, the types of preceding or concomitant treatments, if any, and other individual characteristics of the patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effective amount” also includes an amount of an RNAi agent that produces some desired local or systemic effect at a reasonable benefit/risk ratio applicable to any treatment. iRNA employed in the methods of the present invention may be administered in a sufficient amount to produce a reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human subjects and animal subjects without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means a pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc magnesium, calcium or zinc stearate, or steric acid), or solvent encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject being treated. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such as magnesium state, sodium lauryl sulfate and talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21) polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids (23) serum component, such as serum albumin, HDL and LDL; and (22) other non-toxic compatible substances employed in pharmaceutical formulations.

The term “sample,” as used herein, includes a collection of similar fluids, cells, or tissues isolated from a subject, as well as fluids, cells, or tissues present within a subject. Examples of biological fluids include blood, serum and serosal fluids, plasma, cerebrospinal fluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samples may include samples from tissues, organs or localized regions. For example, samples may be derived from particular organs, parts of organs, or fluids or cells within those organs. In certain embodiments, samples may be derived from the liver (e.g., whole liver or certain segments of liver or certain types of cells in the liver, such as, e.g., hepatocytes). In some embodiments, a “sample derived from a subject” refers to blood or plasma drawn from the subject.

II. iRNAs of the Invention

Described herein are iRNAs which inhibit the expression of an ANGPTL3 gene. In one embodiment, the iRNA agent includes double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of an ANGPTL3 gene in a cell, such as a cell within a subject, e.g., a mammal, such as a human having a disorder of lipid metabolism, e.g., familial hyperlipidemia. The dsRNA includes an antisense strand having a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of an ANGPTL3gene, The region of complementarity is about 30 nucleotides or less in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, or 18 nucleotides or less in length). Upon contact with a cell expressing the ANGPTL3 gene, the iRNA inhibits the expression of the ANGPTL3 gene (e.g., a human, a primate, a non-primate, or a bird ANGPTL3 gene) by at least about 10% as assayed by, for example, a PCR or branched DNA (bDNA)-based method, or by a protein-based method, such as by immunofluorescence analysis, using, for example, Western Blotting or flowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize to form a duplex structure under conditions in which the dsRNA will be used. One strand of a dsRNA (the antisense strand) includes a region of complementarity that is substantially complementary, and generally fully complementary, to a target sequence. The target sequence can be derived from the sequence of an mRNA formed during the expression of an ANGPTL3gene. The other strand (the sense strand) includes a region that is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. As described elsewhere herein and as known in the art, the complementary sequences of a dsRNA can also be contained as self-complementary regions of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs in length, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

Similarly, the region of complementarity to the target sequence is between 15 and 30 nucleotides in length, e.g., between 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated to be part of the invention.

In some embodiments, the dsRNA is between about 15 and about 20 nucleotides in length, or between about 25 and about 30 nucleotides in length. In general, the dsRNA is long enough to serve as a substrate for the Dicer enzyme. For example, it is well known in the art that dsRNAs longer than about 21-23 nucleotides can serve as substrates for Dicer. As the ordinarily skilled person will also recognize, the region of an RNA targeted for cleavage will most often be part of a larger RNA molecule, often an mRNA molecule. Where relevant, a “part” of an mRNA target is a contiguous sequence of an mRNA target of sufficient length to allow it to be a substrate for RNAi-directed cleavage (i.e., cleavage through a RISC pathway).

One of skill in the art will also recognize that the duplex region is a primary functional portion of a dsRNA, e.g., a duplex region of about 9 to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36, 9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34, 12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33, 15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31, 11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Thus, in one embodiment, to the extent that it becomes processed to a functional duplex, of e.g., 15-30 base pairs, that targets a desired RNA for cleavage, an RNA molecule or complex of RNA molecules having a duplex region greater than 30 base pairs is a dsRNA. Thus, an ordinarily skilled artisan will recognize that in one embodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not a naturally occurring miRNA. In another embodiment, an iRNA agent useful to target ANGPTL3 expression is not generated in the target cell by cleavage of a larger dsRNA.

A dsRNA as described herein can further include one or more single-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides. dsRNAs having at least one nucleotide overhang can have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts. A nucleotide overhang can comprise or consist of a nucleotide/nucleoside analog, including a deoxynucleotide/nucleoside. The overhang(s) can be on the sense strand, the antisense strand or any combination thereof. Furthermore, the nucleotide(s) of an overhang can be present on the 5′-end, 3′-end or both ends of either an antisense or sense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc.

iRNA compounds of the invention may be prepared using a two-step procedure. First, the individual strands of the double-stranded RNA molecule are prepared separately. Then, the component strands are annealed. The individual strands of the siRNA compound can be prepared using solution-phase or solid-phase organic synthesis or both. Organic synthesis offers the advantage that the oligonucleotide strands comprising unnatural or modified nucleotides can be easily prepared. Single-stranded oligonucleotides of the invention can be prepared using solution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the invention includes at least two nucleotide sequences, a sense sequence and an anti-sense sequence. The sense strand is selected from the group of sequences provided in Tables 2, 3, 7, 8, 9 and 10, and the corresponding antisense strand of the sense strand is selected from the group of sequences of Tables 2, 3, 7, 8, 9 and 10. In this aspect, one of the two sequences is complementary to the other of the two sequences, with one of the sequences being substantially complementary to a sequence of an mRNA generated in the expression of an ANGPTL3gene. As such, in this aspect, a dsRNA will include two oligonucleotides, where one oligonucleotide is described as the sense strand in Tables 2, 3, 7, 8, 9 and 10, and the second oligonucleotide is described as the corresponding antisense strand of the sense strand in Tables 2, 3, 7, 8, 9 and 10. In one embodiment, the substantially complementary sequences of the dsRNA are contained on separate oligonucleotides. In another embodiment, the substantially complementary sequences of the dsRNA are contained on a single oligonucleotide.

The skilled person is well aware that dsRNAs having a duplex structure of between about 20 and 23 base pairs, e.g., 21, base pairs have been hailed as particularly effective in inducing RNA interference (Elbashir et al., (2001) EMBO J., 20:6877-6888). However, others have found that shorter or longer RNA duplex structures can also be effective (Chu and Rana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226). In the embodiments described above, by virtue of the nature of the oligonucleotide sequences provided in Tables 2, 3, 7, 8, 9 and 10, dsRNAs described herein can include at least one strand of a length of minimally 21 nucleotides. It can be reasonably expected that shorter duplexes having one of the sequences of Tables 2, 3, 7, 8, 9 and 10 minus only a few nucleotides on one or both ends can be similarly effective as compared to the dsRNAs described above. Hence, dsRNAs having a sequence of at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derived from one of the sequences of Tables 2, 3, 7, 8, 9 and 10, and differing in their ability to inhibit the expression of an ANGPTL3gene by not more than about 5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the full sequence, are contemplated to be within the scope of the present invention.

In addition, the RNAs provided in Tables 2, 3, 7, 8, 9 and 10 identify a site(s) in an ANGPTL3 transcript that is susceptible to RISC-mediated cleavage. As such, the present invention further features iRNAs that target within one of these sites. As used herein, an iRNA is said to target within a particular site of an RNA transcript if the iRNA promotes cleavage of the transcript anywhere within that particular site. Such an iRNA will generally include at least about 15 contiguous nucleotides from one of the sequences provided in Tables 2, 3, 7, 8, 9 and 10 coupled to additional nucleotide sequences taken from the region contiguous to the selected sequence in an ANGPTL3gene.

While a target sequence is generally about 15-30 nucleotides in length, there is wide variation in the suitability of particular sequences in this range for directing cleavage of any given target RNA. Various software packages and the guidelines set out herein provide guidance for the identification of optimal target sequences for any given gene target, but an empirical approach can also be taken in which a “window” or “mask” of a given size (as a non-limiting example, 21 nucleotides) is literally or figuratively (including, e.g., in silico) placed on the target RNA sequence to identify sequences in the size range that can serve as target sequences. By moving the sequence “window” progressively one nucleotide upstream or downstream of an initial target sequence location, the next potential target sequence can be identified, until the complete set of possible sequences is identified for any given target size selected. This process, coupled with systematic synthesis and testing of the identified sequences (using assays as described herein or as known in the art) to identify those sequences that perform optimally can identify those RNA sequences that, when targeted with an iRNA agent, mediate the best inhibition of target gene expression. Thus, while the sequences identified, for example, in Tables 2, 3, 7, 8, 9 and 10 represent effective target sequences, it is contemplated that further optimization of inhibition efficiency can be achieved by progressively “walking the window” one nucleotide upstream or downstream of the given sequences to identify sequences with equal or better inhibition characteristics.

Further, it is contemplated that for any sequence identified, e.g., in Tables 2, 3, 7, 8, 9 and 10, further optimization could be achieved by systematically either adding or removing nucleotides to generate longer or shorter sequences and testing those sequences generated by walking a window of the longer or shorter size up or down the target RNA from that point. Again, coupling this approach to generating new candidate targets with testing for effectiveness of iRNAs based on those target sequences in an inhibition assay as known in the art and/or as described herein can lead to further improvements in the efficiency of inhibition. Further still, such optimized sequences can be adjusted by, e.g., the introduction of modified nucleotides as described herein or as known in the art, addition or changes in overhang, or other modifications as known in the art and/or discussed herein to further optimize the molecule (e.g., increasing serum stability or circulating half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to a particular location or cell type, increasing interaction with silencing pathway enzymes, increasing release from endosomes) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to the target sequence. In one embodiment, an iRNA as described herein contains no more than 3 mismatches. If the antisense strand of the iRNA contains mismatches to a target sequence, it is preferable that the area of mismatch is not located in the center of the region of complementarity. If the antisense strand of the iRNA contains mismatches to the target sequence, it is preferable that the mismatch be restricted to be within the last 5 nucleotides from either the 5′- or 3′-end of the region of complementarity. For example, for a 23 nucleotide iRNA agent the strand which is complementary to a region of an ANGPTL3 gene, generally does not contain any mismatch within the central 13 nucleotides. The methods described herein or methods known in the art can be used to determine whether an iRNA containing a mismatch to a target sequence is effective in inhibiting the expression of an ANGPTL3 gene. Consideration of the efficacy of iRNAs with mismatches in inhibiting expression of an ANGPTL3 gene is important, especially if the particular region of complementarity in an ANGPTL3 gene is known to have polymorphic sequence variation within the population.

III. Modified iRNAs of the Invention

In one embodiment, the RNA of an iRNA of the invention, e.g., a dsRNA, is chemically modified to enhance stability or other beneficial characteristics. The nucleic acids featured in the invention can be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Modifications include, for example, end modifications, e.g., 5′-end modifications (phosphorylation, conjugation, inverted linkages) or 3′-end modifications (conjugation, DNA nucleotides, inverted linkages, etc.); base modifications, e.g., replacement with stabilizing bases, destabilizing bases, or bases that base pair with an expanded repertoire of partners, removal of bases (abasic nucleotides), or conjugated bases; sugar modifications (e.g., at the 2′-position or 4′-position) or replacement of the sugar; and/or backbone modifications, including modification or replacement of the phosphodiester linkages. Specific examples of iRNA compounds useful in the embodiments described herein include, but are not limited to RNAs containing modified backbones or no natural internucleoside linkages. RNAs having modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes referenced in the art, modified RNAs that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, a modified iRNA will have a phosphorus atom in its internucleoside backbone.

Modified RNA backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170; 6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423; 6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294; 6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. RE39464, the entire contents of each of which are hereby incorporated herein by reference.

Modified RNA backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatoms and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the above oligonucleosides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 564,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and, 5,677,439, the entire contents of each of which are hereby incorporated herein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use in iRNAs, in which both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an RNA mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of an RNA is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, the entire contents of each of which are hereby incorporated herein by reference. Additional PNA compounds suitable for use in the iRNAs of the invention are described in, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs with phosphorothioate backbones and oligonucleosides with heteroatom backbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known as a methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—, —CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂— [wherein the native phosphodiester backbone is represented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No. 5,489,677, and the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAs featured herein have morpholino backbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties. The iRNAs, e.g., dsRNAs, featured herein can include one of the following at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkyl or C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modifications include O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂, O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where n and m are from 1 to about 10. In other embodiments, dsRNAs include one of the following at the 2′ position: C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of an iRNA, or a group for improving the pharmacodynamic properties of an iRNA, and other substituents having similar properties. In some embodiments, the modification includes a 2′-methoxyethoxy (2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxy group. Another exemplary modification is 2′-dimethylaminooxyethoxy, i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described in examples herein below, and 2′-dimethylaminoethoxyethoxy (also known in the art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e., 2′-O—CH₂—O—CH₂—N(CH₂)₂.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy (2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications can also be made at other positions on the RNA of an iRNA, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which are commonly owned with the instant application. The entire contents of each of the foregoing are hereby incorporated herein by reference.

An iRNA can also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed by Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the invention. These include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, the above noted U.S. Pat. Nos. 3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; 5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and 7,495,088, the entire contents of each of which are hereby incorporated herein by reference.

The RNA of an iRNA can also be modified to include one or more locked nucleic acids (LNA). A locked nucleic acid is a nucleotide having a modified ribose moiety in which the ribose moiety comprises an extra bridge connecting the 2′ and 4′ carbons. This structure effectively “locks” the ribose in the 3′-endo structural conformation. The addition of locked nucleic acids to siRNAs has been shown to increase siRNA stability in serum, and to reduce off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

Representative U.S. Patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207; 7,084,125; and 7,399,845, the entire contents of each of which are hereby incorporated herein by reference.

Potentially stabilizing modifications to the ends of RNA molecules can include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others. Disclosure of this modification can be found in PCT Publication No. WO 2011/005861.

IV. iRNAs Conjugated to Ligands

Another modification of the RNA of an iRNA of the invention involves chemically linking to the RNA one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the iRNA. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al., (1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Res., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993) Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al., (1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g., molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine or hyaluronic acid); or a lipid. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a kidney cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a hepatic cell. Ligands can also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

In some embodiments, a ligand attached to an iRNA as described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophiles, bile acids, steroids, phospholipid analogues, peptides, protein binding agents, PEG, vitamins etc. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc. Oligonucleotides that comprise a number of phosphorothioate linkages are also known to bind to serum protein, thus short oligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases, comprising multiple of phosphorothioate linkages in the backbone are also amenable to the present invention as ligands (e.g. as PK modulating ligands). In addition, aptamers that bind serum components (e.g. serum proteins) are also suitable for use as PK modulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the invention may be synthesized by the use of an oligonucleotide that bears a pendant reactive functionality, such as that derived from the attachment of a linking molecule onto the oligonucleotide (described below). This reactive oligonucleotide may be reacted directly with commercially-available ligands, ligands that are synthesized bearing any of a variety of protecting groups, or ligands that have a linking moiety attached thereto.

The oligonucleotides used in the conjugates of the present invention may be conveniently and routinely made through the well-known technique of solid-phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is also known to use similar techniques to prepare other oligonucleotides, such as the phosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearing sequence-specific linked nucleosides of the present invention, the oligonucleotides and oligonucleosides may be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already bear the linking moiety, ligand-nucleotide or nucleoside-conjugate precursors that already bear the ligand molecule, or non-nucleoside ligand-bearing building blocks.

When using nucleotide-conjugate precursors that already bear a linking moiety, the synthesis of the sequence-specific linked nucleosides is typically completed, and the ligand molecule is then reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the present invention are synthesized by an automated synthesizer using phosphoramidites derived from ligand-nucleoside conjugates in addition to the standard phosphoramidites and non-standard phosphoramidites that are commercially available and routinely used in oligonucleotide synthesis.

A. Lipid Conjugates

In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. For example, the target tissue can be the liver, including parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by target cells such as liver cells. Also included are HSA and low density lipoprotein (LDL).

B. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as tat or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 13). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO: 10) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 11) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 12) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to a dsRNA agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the invention may be linear or cyclic, and may be modified, e.g., glyciosylated or methylated, to facilitate targeting to a specific tissue(s). RGD-containing peptides and peptidiomimemtics may include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the integrin ligand. Preferred conjugates of this ligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, a α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

C. Carbohydrate Conjugates

In some embodiments of the compositions and methods of the invention, an iRNA oligonucleotide further comprises a carbohydrate. The carbohydrate conjugated iRNA are advantageous for the in vivo delivery of nucleic acids, as well as compositions suitable for in vivo therapeutic use, as described herein. As used herein, “carbohydrate” refers to a compound which is either a carbohydrate per se made up of one or more monosaccharide units having at least 6 carbon atoms (which can be linear, branched or cyclic) with an oxygen, nitrogen or sulfur atom bonded to each carbon atom; or a compound having as a part thereof a carbohydrate moiety made up of one or more monosaccharide units each having at least six carbon atoms (which can be linear, branched or cyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include the sugars (mono-, di-, tri- and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9 monosaccharide units), and polysaccharides such as starches, glycogen, cellulose and polysaccharide gums. Specific monosaccharides include C5 and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide. In one embodiment, the monosaccharide is an N-acetylgalactosamine, such as

In another embodiment, a carbohydrate conjugate for use in the compositions and methods of the invention is selected from the group consisting of:

Another representative carbohydrate conjugate for use in the embodiments described herein includes, but is not limited to,

(Formula XXIII), when one of X or Y is an oligonucleotide, the other is a hydrogen.

In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and/or a cell permeation peptide.

D. Linkers

In some embodiments, the conjugate or ligand described herein can be attached to an iRNA oligonucleotide with various linkers that can be cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety that connects two parts of a compound, e.g., covalently attaches two parts of a compound. Linkers typically comprise a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or a chain of atoms, such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylhererocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, which one or more methylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is between about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outside the cell, but which upon entry into a target cell is cleaved to release the two parts the linker is holding together. In a preferred embodiment, the cleavable linking group is cleaved at least about 10 times, 20, times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90 times or more, or at least about 100 times faster in a target cell or under a first reference condition (which can, e.g., be selected to mimic or represent intracellular conditions) than in the blood of a subject, or under a second reference condition (which can, e.g., be selected to mimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH, redox potential or the presence of degradative molecules. Generally, cleavage agents are more prevalent or found at higher levels or activities inside cells than in serum or blood. Examples of such degradative agents include: redox agents which are selected for particular substrates or which have no substrate specificity, including, e.g., oxidative or reductive enzymes or reductive agents such as mercaptans, present in cells, that can degrade a redox cleavable linking group by reduction; esterases; endosomes or agents that can create an acidic environment, e.g., those that result in a pH of five or lower; enzymes that can hydrolyze or degrade an acid cleavable linking group by acting as a general acid, peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptible to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, in the range of 5.5-6.0, and lysosomes have an even more acidic pH at around 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing a cationic lipid from the ligand inside the cell, or into the desired compartment of the cell.

A linker can include a cleavable linking group that is cleavable by a particular enzyme. The type of cleavable linking group incorporated into a linker can depend on the cell to be targeted. For example, a liver-targeting ligand can be linked to a cationic lipid through a linker that includes an ester group. Liver cells are rich in esterases, and therefore the linker will be cleaved more efficiently in liver cells than in cell types that are not esterase-rich. Other cell-types rich in esterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell types rich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability of a degradative agent (or condition) to cleave the candidate linking group. It will also be desirable to also test the candidate cleavable linking group for the ability to resist cleavage in the blood or when in contact with other non-target tissue. Thus, one can determine the relative susceptibility to cleavage between a first and a second condition, where the first is selected to be indicative of cleavage in a target cell and the second is selected to be indicative of cleavage in other tissues or biological fluids, e.g., blood or serum. The evaluations can be carried out in cell free systems, in cells, in cell culture, in organ or tissue culture, or in whole animals. It can be useful to make initial evaluations in cell-free or culture conditions and to confirm by further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of reductively cleavable linking group is a disulphide linking group (—S—S—). To determine if a candidate cleavable linking group is a suitable “reductively cleavable linking group,” or for example is suitable for use with a particular iRNA moiety and particular targeting agent one can look to methods described herein. For example, a candidate can be evaluated by incubation with dithiothreitol (DTT), or other reducing agent using reagents know in the art, which mimic the rate of cleavage which would be observed in a cell, e.g., a target cell. The candidates can also be evaluated under conditions which are selected to mimic blood or serum conditions. In one, candidate compounds are cleaved by at most about 10% in the blood. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in the cell (or under in vitro conditions selected to mimic intracellular conditions) as compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The rate of cleavage of candidate compounds can be determined using standard enzyme kinetics assays under conditions chosen to mimic intracellular media and compared to conditions chosen to mimic extracellular media.

ii. Phosphate-Based Cleavable Linking Groups

In another embodiment, a cleavable linker comprises a phosphate-based cleavable linking group. A phosphate-based cleavable linking group is cleaved by agents that degrade or hydrolyze the phosphate group. An example of an agent that cleaves phosphate groups in cells are enzymes such as phosphatases in cells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—, —O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—, —S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—, —O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—, —O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—, —O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—, —S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—, —O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—, —O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. These candidates can be evaluated using methods analogous to those described above.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In preferred embodiments acid cleavable linking groups are cleaved in an acidic environment with a pH of about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower), or by agents such as enzymes that can act as a general acid. In a cell, specific low pH organelles, such as endosomes and lysosomes can provide a cleaving environment for acid cleavable linking groups. Examples of acid cleavable linking groups include but are not limited to hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is when the carbon attached to the oxygen of the ester (the alkoxy group) is an aryl group, substituted alkyl group, or tertiary alkyl group such as dimethyl pentyl or t-butyl. These candidates can be evaluated using methods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-based cleavable linking group. An ester-based cleavable linking group is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable linking groups include but are not limited to esters of alkylene, alkenylene and alkynylene groups. Ester cleavable linking groups have the general formula —C(O)O—, or —OC(O)—. These candidates can be evaluated using methods analogous to those described above.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-based cleavable linking group. A peptide-based cleavable linking group is cleaved by enzymes such as peptidases and proteases in cells. Peptide-based cleavable linking groups are peptide bonds formed between amino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides. Peptide-based cleavable groups do not include the amide group (—C(O)NH—). The amide group can be formed between any alkylene, alkenylene or alkynelene. A peptide bond is a special type of amide bond formed between amino acids to yield peptides and proteins. The peptide based cleavage group is generally limited to the peptide bond (i.e., the amide bond) formed between amino acids yielding peptides and proteins and does not include the entire amide functional group. Peptide-based cleavable linking groups have the general formula —NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the two adjacent amino acids. These candidates can be evaluated using methods analogous to those described above.

In one embodiment, an iRNA of the invention is conjugated to a carbohydrate through a linker. Non-limiting examples of iRNA carbohydrate conjugates with linkers of the compositions and methods of the invention include, but are not limited to,

when one of X or Y is an oligonucleotide, the other is a hydrogen.

In certain embodiments of the compositions and methods of the invention, a ligand is one or more GalNAc (N-acetylgalactosamine) derivatives attached through a bivalent or trivalent branched linker.

In one embodiment, a dsRNA of the invention is conjugated to a bivalent or trivalent branched linker selected from the group of structures shown in any of formula (XXXI)-(XXXIV):

wherein: q2A, q2B, q3A, q3B, q4A, q4B, q5A, q5B and q5C represent independently for each occurrence 0-20 and wherein the repeating unit can be the same or different; P^(2A), P^(2B), P^(3A), P^(3B), P^(4A), P^(4B), P^(5A), P^(5B), P^(5C), T^(2A), T^(2B), T^(3A), T^(3B), T^(4A), T^(4B), T^(4A), T^(5B), T^(5C) are each independently for each occurrence absent, CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH or CH₂O; Q^(2A), Q^(2B), Q^(3A), Q^(3B), Q^(4A), Q^(4B), Q^(5A), Q^(5B), Q^(5C) are independently for each occurrence absent, alkylene, substituted alkylene wherein one or more methylenes can be interrupted or terminated by one or more of O, S, S(O), SO₂, N(R^(N)), C(R′)═C(R″), C≡C or C(O); R^(2A), R^(2B), R^(3A), R^(3B), R^(4A), R^(4B), R^(5A), R^(5B), R^(5C) are each independently for each occurrence absent, NH, O, S, CH₂, C(O)O, C(O)NH, NHCH(R^(a))C(O), —C(O)—CH(R^(a))—NH—, CO,

or heterocyclyl;

L^(2A), L^(2B), L^(3A), L^(3B), L^(4A), L^(4B), L^(5A), L^(5B) and L^(5C) represent the ligand; i.e. each independently for each occurrence a monosaccharide (such as GalNAc), disaccharide, trisaccharide, tetrasaccharide, oligosaccharide, or polysaccharide; and R^(a) is H or amino acid side chain. Trivalent conjugating GalNAc derivatives are particularly useful for use with RNAi agents for inhibiting the expression of a target gene, such as those of formula (XXXV):

-   -   wherein L^(5A), L^(5B) and L^(5C) represent a monosaccharide,         such as GalNAc derivative.

Examples of suitable bivalent and trivalent branched linker groups conjugating GalNAc derivatives include, but are not limited to, the structures recited above as formulas II_VII, XI, X, and XIII.

Representative U.S. patents that teach the preparation of RNA conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931; 6,900,297; 7,037,646; 8,106,022, the entire contents of each of which are hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated in a single compound or even at a single nucleoside within an iRNA. The present invention also includes iRNA compounds that are chimeric compounds.

“Chimeric” iRNA compounds or “chimeras,” in the context of this invention, are iRNA compounds, preferably dsRNAs, which contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of a dsRNA compound. These iRNAs typically contain at least one region wherein the RNA is modified so as to confer upon the iRNA increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the iRNA can serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of iRNA inhibition of gene expression. Consequently, comparable results can often be obtained with shorter iRNAs when chimeric dsRNAs are used, compared to phosphorothioate deoxy dsRNAs hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligand group. A number of non-ligand molecules have been conjugated to iRNAs in order to enhance the activity, cellular distribution or cellular uptake of the iRNA, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties have included lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative United States patents that teach the preparation of such RNA conjugates have been listed above. Typical conjugation protocols involve the synthesis of an RNAs bearing an aminolinker at one or more positions of the sequence. The amino group is then reacted with the molecule being conjugated using appropriate coupling or activating reagents. The conjugation reaction can be performed either with the RNA still bound to the solid support or following cleavage of the RNA, in solution phase. Purification of the RNA conjugate by HPLC typically affords the pure conjugate.

IV. Delivery of an iRNA of the Invention

The delivery of an iRNA of the invention to a cell e.g., a cell within a subject, such as a human subject (e.g., a subject in need thereof, such as a subject having a disorder of lipid metabolism) can be achieved in a number of different ways. For example, delivery may be performed by contacting a cell with an iRNA of the invention either in vitro or in vivo. In vivo delivery may also be performed directly by administering a composition comprising an iRNA, e.g., a dsRNA, to a subject. Alternatively, in vivo delivery may be performed indirectly by administering one or more vectors that encode and direct the expression of the iRNA. These alternatives are discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitro or in vivo) can be adapted for use with an iRNA of the invention (see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporated herein by reference in their entireties). For in vivo delivery, factors to consider in order to deliver an iRNA molecule include, for example, biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. The non-specific effects of an iRNA can be minimized by local administration, for example, by direct injection or implantation into a tissue or topically administering the preparation. Local administration to a treatment site maximizes local concentration of the agent, limits the exposure of the agent to systemic tissues that can otherwise be harmed by the agent or that can degrade the agent, and permits a lower total dose of the iRNA molecule to be administered. Several studies have shown successful knockdown of gene products when an iRNA is administered locally. For example, intraocular delivery of a VEGF dsRNA by intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al., (2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J. et al. (2003) Mol. Vis. 9:210-216) were both shown to prevent neovascularization in an experimental model of age-related macular degeneration. In addition, direct intratumoral injection of a dsRNA in mice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther. 11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. et al., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther. 15:515-523). RNA interference has also shown success with local delivery to the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids 32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al. (2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience 129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602) and to the lungs by intranasal administration (Howard, K A. et al., (2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem. 279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). For administering an iRNA systemically for the treatment of a disease, the RNA can be modified or alternatively delivered using a drug delivery system; both methods act to prevent the rapid degradation of the dsRNA by endo- and exo-nucleases in vivo. Modification of the RNA or the pharmaceutical carrier can also permit targeting of the iRNA composition to the target tissue and avoid undesirable off-target effects. iRNA molecules can be modified by chemical conjugation to lipophilic groups such as cholesterol to enhance cellular uptake and prevent degradation. For example, an iRNA directed against ApoB conjugated to a lipophilic cholesterol moiety was injected systemically into mice and resulted in knockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. et al., (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer has been shown to inhibit tumor growth and mediate tumor regression in a mouse model of prostate cancer (McNamara, J O. et al., (2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can be delivered using drug delivery systems such as a nanoparticle, a dendrimer, a polymer, liposomes, or a cationic delivery system. Positively charged cationic delivery systems facilitate binding of an iRNA molecule (negatively charged) and also enhance interactions at the negatively charged cell membrane to permit efficient uptake of an iRNA by the cell. Cationic lipids, dendrimers, or polymers can either be bound to an iRNA, or induced to form a vesicle or micelle (see e.g., Kim S H. et al., (2008) Journal of Controlled Release 129(2):107-116) that encases an iRNA. The formation of vesicles or micelles further prevents degradation of the iRNA when administered systemically. Methods for making and administering cationic-iRNA complexes are well within the abilities of one skilled in the art (see e.g., Sorensen, D R., et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, A S et al., (2007) J. Hypertens. 25:197-205, which are incorporated herein by reference in their entirety). Some non-limiting examples of drug delivery systems useful for systemic delivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solid nucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epub ahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA forms a complex with cyclodextrin for systemic administration. Methods for administration and pharmaceutical compositions of iRNAs and cyclodextrins can be found in U.S. Pat. No. 7,427,605, which is herein incorporated by reference in its entirety.

A. Vector Encoded iRNAs of the Invention

iRNA targeting the ANGPTL3 gene can be expressed from transcription units inserted into DNA or RNA vectors (see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., et al., International PCT Publication No. WO 00/22113, Conrad, International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can be transient (on the order of hours to weeks) or sustained (weeks to months or longer), depending upon the specific construct used and the target tissue or cell type. These transgenes can be introduced as a linear construct, a circular plasmid, or a viral vector, which can be an integrating or non-integrating vector. The transgene can also be constructed to permit it to be inherited as an extrachromosomal plasmid (Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of an iRNA can be transcribed from a promoter on an expression vector. Where two separate strands are to be expressed to generate, for example, a dsRNA, two separate expression vectors can be co-introduced (e.g., by transfection or infection) into a target cell. Alternatively each individual strand of a dsRNA can be transcribed by promoters both of which are located on the same expression plasmid. In one embodiment, a dsRNA is expressed as inverted repeat polynucleotides joined by a linker polynucleotide sequence such that the dsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors. Expression vectors compatible with eukaryotic cells, preferably those compatible with vertebrate cells, can be used to produce recombinant constructs for the expression of an iRNA as described herein. Eukaryotic cell expression vectors are well known in the art and are available from a number of commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired nucleic acid segment. Delivery of iRNA expressing vectors can be systemic, such as by intravenous or intramuscular administration, by administration to target cells ex-planted from the patient followed by reintroduction into the patient, or by any other means that allows for introduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as a complex with cationic lipid carriers (e.g., Oligofectamine) or non-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipid transfections for iRNA-mediated knockdowns targeting different regions of a target RNA over a period of a week or more are also contemplated by the invention. Successful introduction of vectors into host cells can be monitored using various known methods. For example, transient transfection can be signaled with a reporter, such as a fluorescent marker, such as Green Fluorescent Protein (GFP). Stable transfection of cells ex vivo can be ensured using markers that provide the transfected cell with resistance to specific environmental factors (e.g., antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods and compositions described herein include, but are not limited to, (a) adenovirus vectors; (b) retrovirus vectors, including but not limited to lentiviral vectors, moloney murine leukemia virus, etc.; (c) adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV 40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h) picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g., vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) a helper-dependent or gutless adenovirus. Replication-defective viruses can also be advantageous. Different vectors will or will not become incorporated into the cells' genome. The constructs can include viral sequences for transfection, if desired. Alternatively, the construct can be incorporated into vectors capable of episomal replication, e.g. EPV and EBV vectors. Constructs for the recombinant expression of an iRNA will generally require regulatory elements, e.g., promoters, enhancers, etc., to ensure the expression of the iRNA in target cells. Other aspects to consider for vectors and constructs are further described below.

Vectors useful for the delivery of an iRNA will include regulatory elements (promoter, enhancer, etc.) sufficient for expression of the iRNA in the desired target cell or tissue. The regulatory elements can be chosen to provide either constitutive or regulated/inducible expression.

Expression of the iRNA can be precisely regulated, for example, by using an inducible regulatory sequence that is sensitive to certain physiological regulators, e.g., circulating glucose levels, or hormones (Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expression systems, suitable for the control of dsRNA expression in cells or in mammals include, for example, regulation by ecdysone, by estrogen, progesterone, tetracycline, chemical inducers of dimerization, and isopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in the art would be able to choose the appropriate regulatory/promoter sequence based on the intended use of the iRNA transgene.

Viral vectors that contain nucleic acid sequences encoding an iRNA can be used. For example, a retroviral vector can be used (see Miller et al., (1993) Meth. Enzymol. 217:581-599). These retroviral vectors contain the components necessary for the correct packaging of the viral genome and integration into the host cell DNA. The nucleic acid sequences encoding an iRNA are cloned into one or more vectors, which facilitate delivery of the nucleic acid into a patient. More detail about retroviral vectors can be found, for example, in Boesen et al., Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdr1 gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy. Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al., (1994) J. Clin. Invest. 93:644-651; Kiem et al., (1994) Blood 83:1467-1473; Salmons and Gunzberg, (1993) Human Gene Therapy 4:129-141; and Grossman and Wilson, (1993) Curr. Opin. in Genetics and Devel. 3:110-114. Lentiviral vectors contemplated for use include, for example, the HIV based vectors described in U.S. Pat. Nos. 6,143,520; 5,665,557; and 5,981,276, which are herein incorporated by reference.

Adenoviruses are also contemplated for use in delivery of iRNAs of the invention. Adenoviruses are especially attractive vehicles, e.g., for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, (1993) Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al., (1994) Human Gene Therapy 5:3-10 demonstrated the use of adenovirus vectors to transfer genes to the respiratory epithelia of rhesus monkeys. Other instances of the use of adenoviruses in gene therapy can be found in Rosenfeld et al., (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell 68:143-155; Mastrangeli et al., (1993) J. Clin. Invest. 91:225-234; PCT Publication WO94/12649; and Wang et al., (1995) Gene Therapy 2:775-783. A suitable AV vector for expressing an iRNA featured in the invention, a method for constructing the recombinant AV vector, and a method for delivering the vector into target cells, are described in Xia H et al. (2002), Nat. Biotech. 20: 1006-1010.

Adeno-associated virus (AAV) vectors may also be used to delivery an iRNA of the invention (Walsh et al., (1993) Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436,146). In one embodiment, the iRNA can be expressed as two separate, complementary single-stranded RNA molecules from a recombinant AAV vector having, for example, either the U6 or H1 RNA promoters, or the cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressing the dsRNA featured in the invention, methods for constructing the recombinant AV vector, and methods for delivering the vectors into target cells are described in Samulski R et al. (1987), J. Virol. 61: 3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski R et al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. Nos. 5,252,479; 5,139,941; International Patent Application No. WO 94/13788; and International Patent Application No. WO 93/24641, the entire disclosures of which are herein incorporated by reference.

Another viral vector suitable for delivery of an iRNA of the invention is a pox virus such as a vaccinia virus, for example an attenuated vaccinia such as Modified Virus Ankara (MVA) or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectors with envelope proteins or other surface antigens from other viruses, or by substituting different viral capsid proteins, as appropriate. For example, lentiviral vectors can be pseudotyped with surface proteins from vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and the like. AAV vectors can be made to target different cells by engineering the vectors to express different capsid protein serotypes; see, e.g., Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosure of which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in an acceptable diluent, or can include a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells which produce the gene delivery system.

V. Pharmaceutical Compositions of the Invention

The present invention also includes pharmaceutical compositions and formulations which include the iRNAs of the invention. In one embodiment, provided herein are pharmaceutical compositions containing an iRNA, as described herein, and a pharmaceutically acceptable carrier. The pharmaceutical compositions containing the iRNA are useful for treating a disease or disorder associated with the expression or activity of an ANGPTL3 gene, e.g., a disorder of lipid metabolism, such as hypertriglyceridemia.

Such pharmaceutical compositions are formulated based on the mode of delivery. One example is compositions that are formulated for systemic administration via parenteral delivery, e.g., by intravenous (IV) or for subcutaneous delivery. Another example is compositions that are formulated for direct delivery into the liver, e.g., by infusion into the liver, such as by continuous pump infusion.

The pharmaceutical compositions of the invention may be administered in dosages sufficient to inhibit expression of a ANGPTL3 gene. In general, a suitable dose of an iRNA of the invention will be in the range of about 0.001 to about 200.0 milligrams per kilogram body weight of the recipient per day, generally in the range of about 1 to 50 mg per kilogram body weight per day. For example, the dsRNA can be administered at about 0.01 mg/kg, about 0.05 mg/kg, about 0.5 mg/kg, about 1 mg/kg, about 1.5 mg/kg, about 2 mg/kg, about 3 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, or about 50 mg/kg per single dose.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In another embodiment, the dsRNA is administered at a dose of about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of iRNA, such as about 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

The pharmaceutical composition can be administered once daily, or the iRNA can be administered as two, three, or more sub-doses at appropriate intervals throughout the day or even using continuous infusion or delivery through a controlled release formulation. In that case, the iRNA contained in each sub-dose must be correspondingly smaller in order to achieve the total daily dosage. The dosage unit can also be compounded for delivery over several days, e.g., using a conventional sustained release formulation which provides sustained release of the iRNA over a several day period. Sustained release formulations are well known in the art and are particularly useful for delivery of agents at a particular site, such as could be used with the agents of the present invention. In this embodiment, the dosage unit contains a corresponding multiple of the daily dose.

The effect of a single dose on ANGPTL3 levels can be long lasting, such that subsequent doses are administered at not more than 3, 4, or 5 day intervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors can influence the dosage and timing required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. Estimates of effective dosages and in vivo half-lives for the individual iRNAs encompassed by the invention can be made using conventional methodologies or on the basis of in vivo testing using an appropriate animal model, as described elsewhere herein.

Advances in mouse genetics have generated a number of mouse models for the study of various human diseases, such as disorders of lipid metabolism that would benefit from reduction in the expression of ANGPTL3. Such models can be used for in vivo testing of iRNA, as well as for determining a therapeutically effective dose. Suitable mouse models are known in the art and include, for example, an obese (ob/ob) mouse containing a mutation in the obese (ob) gene (Wiegman et al., (2003) Diabetes, 52:1081-1089); a mouse containing homozygous knock-out of an LDL receptor (LDLR−/− mouse; Ishibashi et al., (1993) J Clin Invest 92(2):883-893); diet-induced artherosclerosis mouse model (Ishida et al., (1991) J. Lipid. Res., 32:559-568); and heterozygous lipoprotein lipase knockout mouse model (Weistock et al., (1995) J. Clin. Invest. 96(6):2555-2568).

The pharmaceutical compositions of the present invention can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be topical (e.g., by a transdermal patch), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal, oral or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; subdermal, e.g., via an implanted device; or intracranial, e.g., by intraparenchymal, intrathecal or intraventricular, administration.

The iRNA can be delivered in a manner to target a particular tissue, such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administration can include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like can be necessary or desirable. Coated condoms, gloves and the like can also be useful. Suitable topical formulations include those in which the iRNAs featured in the invention are in admixture with a topical delivery agent such as lipids, liposomes, fatty acids, fatty acid esters, steroids, chelating agents and surfactants. Suitable lipids and liposomes include neutral (e.g., dioleoylphosphatidyl DOPE ethanolamine, dimyristoylphosphatidyl choline DMPC, distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidyl glycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAP and dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in the invention can be encapsulated within liposomes or can form complexes thereto, in particular to cationic liposomes. Alternatively, iRNAs can be complexed to lipids, in particular to cationic lipids. Suitable fatty acids and esters include but are not limited to arachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride, diglyceride or pharmaceutically acceptable salt thereof. Topical formulations are described in detail in U.S. Pat. No. 6,747,014, which is incorporated herein by reference.

A. iRNA Formulations Comprising Membranous Molecular Assemblies

An iRNA for use in the compositions and methods of the invention can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety of methods. In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAi agent preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the RNAi agent and condense around the RNAi agent to form a liposome. After condensation, the detergent is removed, e.g., by dialysis, to yield a liposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are further described in, e.g., WO 96/37194, the entire contents of which are incorporated herein by reference. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Bangham et al., (1965) M. Mol. Biol. 23:238; Olson et al., (1979) Biochim. Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75: 4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al., (1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161. Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. These methods are readily adapted to packaging RNAi agent preparations into liposomes.

Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged nucleic acid molecules to form a stable complex. The positively charged nucleic acid/liposome complex binds to the negatively charged cell surface and is internalized in an endosome. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleic acids rather than complex with them. Since both the nucleic acid and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid is entrapped within the aqueous interior of these liposomes. pH sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J. 11:417.

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome™ I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver cyclosporin-A into the dermis of mouse skin. Results indicated that such non-ionic liposomal systems were effective in facilitating the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more glycolipids, such as monosialoganglioside G_(M1), or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64) reported the ability of monosialoganglioside G_(M1), galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al., disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Lim et al).

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated RNAi agents in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of RNAi agent (see, e.g., Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer RNAi agent into the skin. In some implementations, liposomes are used for delivering RNAi agent to epidermal cells and also to enhance the penetration of RNAi agent into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with RNAi agent are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include RNAi agent can be delivered, for example, subcutaneously by infection in order to deliver RNAi agent to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading.

Other formulations amenable to the present invention are described in U.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008; 61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008; 61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCT application no PCT/US2007/080331, filed Oct. 3, 2007 also describes formulations that are amenable to the present invention.

Transfersomes are yet another type of liposomes, and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the skin), self-repairing, frequently reach their targets without fragmenting, and often self-loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the “head”) provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

The iRNA for use in the methods of the invention can also be provided as micellar formulations. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the siRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the siRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the siRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e., there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g., through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. In certain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g., at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

B. Nucleic Acid Lipid Particles

iRNAs, e.g., dsRNAs of in the invention may be fully encapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP, SNALP, or other nucleic acid-lipid particle. As used herein, the term “SNALP” refers to a stable nucleic acid-lipid particle, including SPLP. As used herein, the term “SPLP” refers to a nucleic acid-lipid particle comprising plasmid DNA encapsulated within a lipid vesicle. SNALPs and SPLPs typically contain a cationic lipid, a non-cationic lipid, and a lipid that prevents aggregation of the particle (e.g., a PEG-lipid conjugate). SNALPs and SPLPs are extremely useful for systemic applications, as they exhibit extended circulation lifetimes following intravenous (i.v.) injection and accumulate at distal sites (e.g., sites physically separated from the administration site). SPLPs include “pSPLP,” which include an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 00/03683. The particles of the present invention typically have a mean diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, most typically about 70 nm to about 90 nm, and are substantially nontoxic. In addition, the nucleic acids when present in the nucleic acid-lipid particles of the present invention are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S. Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g., lipid to dsRNA ratio) will be in the range of from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 to about 9:1. Ranges intermediate to the above recited ranges are also contemplated to be part of the invention.

The cationic lipid can be, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanedio (DOAP), 1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (Tech GO, or a mixture thereof. The cationic lipid can comprise from about 20 mol % to about 50 mol % or about 40 mol % of the total lipid present in the particle.

In another embodiment, the compound 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used to prepare lipid-siRNA nanoparticles. Synthesis of 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S. provisional patent application No. 61/107,998 filed on Oct. 23, 2008, which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40% 2, 2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40% Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of 63.0±20 nm and a 0.027 siRNA/Lipid Ratio. The ionizable/non-cationic lipid can be an anionic lipid or a neutral lipid including, but not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. The non-cationic lipid can be from about 5 mol % to about 90 mol %, about 10 mol %, or about 58 mol % if cholesterol is included, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles can be, for example, a polyethyleneglycol (PEG)-lipid including, without limitation, a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), a PEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. The PEG-DAA conjugate can be, for example, a PEG-dilauryloxypropyl (Ci₂), a PEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or a PEG-distearyloxypropyl (C]₈). The conjugated lipid that prevents aggregation of particles can be from 0 mol % to about 20 mol % or about 2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includes cholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol % of the total lipid present in the particle.

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patent application Ser. No. 12/056,230, filed Mar. 26, 2008, which is incorporated herein by reference), Cholesterol (Sigma-Aldrich), and PEG-Ceramide C16 (Avanti Polar Lipids) can be used to prepare lipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions of each in ethanol can be prepared as follows: ND98, 133 mg/ml; Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98, Cholesterol, and PEG-Ceramide C16 stock solutions can then be combined in a, e.g., 42:48:10 molar ratio. The combined lipid solution can be mixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that the final ethanol concentration is about 35-45% and the final sodium acetate concentration is about 100-300 mM. Lipid-dsRNA nanoparticles typically form spontaneously upon mixing. Depending on the desired particle size distribution, the resultant nanoparticle mixture can be extruded through a polycarbonate membrane (e.g., 100 nm cut-off) using, for example, a thermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). In some cases, the extrusion step can be omitted. Ethanol removal and simultaneous buffer exchange can be accomplished by, for example, dialysis or tangential flow filtration. Buffer can be exchanged with, for example, phosphate buffered saline (PBS) at about pH 7, e.g., about pH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or about pH 7.4.

LNP01 formulations are described, e.g., in International Application Publication No. WO 2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are described in the table below.

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugate Ionizable/Cationic Lipid Lipid:siRNA ratio SNALP-1 1,2-Dilinolenyloxy-N,N-dimethylaminopropane DLinDMA/DPPC/Cholesterol/PEG- (DLinDMA) cDMA (57.1/7.1/34.4/1.4) lipid:siRNA ~7:1 2-XTC 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DPPC/Cholesterol/PEG- dioxolane (XTC) cDMA 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP05 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP06 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP07 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP08 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP09 2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]- XTC/DSPC/Cholesterol/PEG-DMG dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10 (3aR,5s,6aS)-N,N-dimethyl-2,2-di((9Z,12Z)- ALN100/DSPC/Cholesterol/PEG- octadeca-9,12-dienyl)tetrahydro-3aH- DMG cyclopenta[d][1,3]dioxol-5-amine (ALN100) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP11 (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31- MC-3/DSPC/Cholesterol/PEG- tetraen-19-yl 4-(dimethylamino)butanoate DMG (MC3) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- Tech G1/DSPC/Cholesterol/PEG- hydroxydodecyl)amino)ethyl)(2- DMG hydroxydodecyl)amino)ethyl)piperazin-1- 50/10/38.5/1.5 yl)ethylazanediyl)didodecan-2-ol (Tech G1) Lipid:siRNA 10:1 LNP13 XTC XTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3 MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3 MC3/DSPC/Chol/PEG- DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1 LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17 MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3 MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200 C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTC XTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC: distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholine PEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avg mol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18) (PEG with avg mol wt of 2000) PEG-cDMA: PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000) SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprising formulations are described in International Publication No. WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated by reference. XTC comprising formulations are described, e.g., in U.S. Provisional Ser. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No. 61/156,851, filed Mar. 2, 2009; U.S. Provisional Ser. No. filed Jun. 10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009; U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, and International Application No. PCT/US2010/022614, filed Jan. 29, 2010, which are hereby incorporated by reference. MC3 comprising formulations are described, e.g., in U.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entire contents of which are hereby incorporated by reference. ALNY-100 comprising formulations are described, e.g., International patent application number PCT/US09/63933, filed on Nov. 10, 2009, which is hereby incorporated by reference. C12-200 comprising formulations are described in U.S. Provisional Ser. No. 61/175,770, filed May 5, 2009 and International Application No. PCT/US10/33777, filed May 5, 2010, which are hereby incorporated by reference.

Synthesis of Ionizable/Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in the nucleic acid-lipid particles of the invention can be prepared by known organic synthesis techniques, including the methods described in more detail in the Examples. All substituents are as defined below unless indicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic, saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms. Representative saturated straight chain alkyls include methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturated branched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl, isopentyl, and the like. Representative saturated cyclic alkyls include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; while unsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, and the like.

“Alkenyl” means an alkyl, as defined above, containing at least one double bond between adjacent carbon atoms. Alkenyls include both cis and trans isomers. Representative straight chain and branched alkenyls include ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl, 1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl, 2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, which additionally contains at least one triple bond between adjacent carbons. Representative straight chain and branched alkynyls include acetylenyl, propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1 butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at the point of attachment is substituted with an oxo group, as defined below. For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acyl groups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-membered bicyclic, heterocyclic ring which is either saturated, unsaturated, or aromatic, and which contains from 1 or 2 heteroatoms independently selected from nitrogen, oxygen and sulfur, and wherein the nitrogen and sulfur heteroatoms can be optionally oxidized, and the nitrogen heteroatom can be optionally quaternized, including bicyclic rings in which any of the above heterocycles are fused to a benzene ring. The heterocycle can be attached via any heteroatom or carbon atom. Heterocycles include heteroaryls as defined below. Heterocycles include morpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl, hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl, tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl, tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substituted alkenyl”, “optionally substituted alkynyl”, “optionally substituted acyl”, and “optionally substituted heterocycle” means that, when substituted, at least one hydrogen atom is replaced with a substituent. In the case of an oxo substituent (═O) two hydrogen atoms are replaced. In this regard, substituents include oxo, halogen, heterocycle, —CN, —ORx, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy, wherein n is 0, 1 or 2, Rx and Ry are the same or different and independently hydrogen, alkyl or heterocycle, and each of said alkyl and heterocycle substituents can be further substituted with one or more of oxo, halogen, —OH, —CN, alkyl, —ORx, heterocycle, —NRxRy, —NRxC(═O)Ry, —NRxSO2Ry, —C(═O)Rx, —C(═O)ORx, —C(═O)NRxRy, —SOnRx and —SOnNRxRy.

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention can require the use of protecting groups. Protecting group methodology is well known to those skilled in the art (see, for example, Protective Groups in Organic Synthesis, Green, T. W. et al., Wiley-Interscience, New York City, 1999). Briefly, protecting groups within the context of this invention are any group that reduces or eliminates unwanted reactivity of a functional group. A protecting group can be added to a functional group to mask its reactivity during certain reactions and then removed to reveal the original functional group. In some embodiments an “alcohol protecting group” is used. An “alcohol protecting group” is any group which decreases or eliminates unwanted reactivity of an alcohol functional group. Protecting groups can be added and removed using techniques well known in the art.

Synthesis of Formula A

In some embodiments, nucleic acid-lipid particles of the invention are formulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring. In some embodiments, the cationic lipid is XTC (2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, the lipid of formula A above can be made by the following Reaction Schemes 1 or 2, wherein all substituents are as defined above unless indicated otherwise.

Lipid A, where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can be optionally substituted, and R3 and R4 are independently lower alkyl or R3 and R4 can be taken together to form an optionally substituted heterocyclic ring, can be prepared according to Scheme 1. Ketone 1 and bromide 2 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 1 and 2 yields ketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A. The lipids of formula A can be converted to the corresponding ammonium salt with an organic salt of formula 5, where X is anion counter ion selected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared according to Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased or prepared according to methods known to those of ordinary skill in the art. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to the corresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e., (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate) was as follows. A solution of (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g), 4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g), 4-N,N-dimethylaminopyridine (0.61 g) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) in dichloromethane (5 mL) was stirred at room temperature overnight. The solution was washed with dilute hydrochloric acid followed by dilute aqueous sodium bicarbonate. The organic fractions were dried over anhydrous magnesium sulphate, filtered and the solvent removed on a rotovap. The residue was passed down a silica gel column (20 g) using a 1-5% methanol/dichloromethane elution gradient. Fractions containing the purified product were combined and the solvent removed, yielding a colorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the following scheme 3:

Synthesis of 515

To a stirred suspension of LiAlH₄ (3.74 g, 0.09852 mol) in 200 ml anhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10 g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere. After complete addition, reaction mixture was warmed to room temperature and then heated to reflux for 4 h. Progress of the reaction was monitored by TLC. After completion of reaction (by TLC) the mixture was cooled to 0° C. and quenched with careful addition of saturated Na₂SO₄ solution. Reaction mixture was stirred for 4 h at room temperature and filtered off. Residue was washed well with THF. The filtrate and washings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCl and stirred for 20 minutes at room temperature. The volatilities were stripped off under vacuum to furnish the hydrochloride salt of 515 as a white solid. Yield: 7.12 g ¹H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL two neck RBF, was added NEt₃ (37.2 mL, 0.2669 mol) and cooled to 0° C. under nitrogen atmosphere. After a slow addition of N-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dry DCM, reaction mixture was allowed to warm to room temperature. After completion of the reaction (2-3 h by TLC) mixture was washed successively with 1N HCl solution (1×100 mL) and saturated NaHCO₃ solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4 and the solvent was evaporated to give crude material which was purified by silica gel column chromatography to get 516 as sticky mass. Yield: 11 g (89%). ¹H-NMR (CDCl₃, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H), 5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m, 2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of 220 mL acetone and water (10:1) in a single neck 500 mL RBF and to it was added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by 4.2 mL of 7.6% solution of OsO₄ (0.275 g, 0.00108 mol) in tert-butanol at room temperature. After completion of the reaction (˜3 h), the mixture was quenched with addition of solid Na₂SO₃ and resulting mixture was stirred for 1.5 h at room temperature. Reaction mixture was diluted with DCM (300 mL) and washed with water (2×100 mL) followed by saturated NaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (lx 50 mL). Organic phase was dried over an.Na₂SO₄ and solvent was removed in vacuum. Silica gel column chromatographic purification of the crude material was afforded a mixture of diastereomers, which were separated by prep HPLC. Yield:—6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz): δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H), 3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3, [M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518

Using a procedure analogous to that described for the synthesis of compound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil. 1H-NMR (CDCl₃, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H), 5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H), 2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H), 1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519

A solution of compound 518 (1 eq) in hexane (15 mL) was added in a drop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq). After complete addition, the mixture was heated at 40° C. over 0.5 h then cooled again on an ice bath. The mixture was carefully hydrolyzed with saturated aqueous Na₂SO₄ then filtered through celite and reduced to an oil. Column chromatography provided the pure 519 (1.3 g, 68%) which was obtained as a colorless oil. ¹³C NMR δ=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight for C₄₄H₈₀NO₂ (M+H)+ Calc. 654.6, Found 654.6.

Formulations prepared by either the standard or extrusion-free method can be characterized in similar manners. For example, formulations are typically characterized by visual inspection. They should be whitish translucent solutions free from aggregates or sediment. Particle size and particle size distribution of lipid-nanoparticles can be measured by light scattering using, for example, a Malvern Zetasizer Nano ZS (Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nm in size. The particle size distribution should be unimodal. The total dsRNA concentration in the formulation, as well as the entrapped fraction, is estimated using a dye exclusion assay. A sample of the formulated dsRNA can be incubated with an RNA-binding dye, such as Ribogreen (Molecular Probes) in the presence or absence of a formulation disrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in the formulation can be determined by the signal from the sample containing the surfactant, relative to a standard curve. The entrapped fraction is determined by subtracting the “free” dsRNA content (as measured by the signal in the absence of surfactant) from the total dsRNA content. Percent entrapped dsRNA is typically >85%. For SNALP formulation, the particle size is at least 30 nm, at least 40 nm, at least 50 nm, at least 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least 100 nm, at least 110 nm, and at least 120 nm. The suitable range is typically about at least 50 nm to about at least 110 nm, about at least 60 nm to about at least 100 nm, or about at least 80 nm to about at least 90 nm.

Compositions and formulations for oral administration include powders or granules, microparticulates, nanoparticulates, suspensions or solutions in water or non-aqueous media, capsules, gel capsules, sachets, tablets or minitablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders can be desirable. In some embodiments, oral formulations are those in which dsRNAs featured in the invention are administered in conjunction with one or more penetration enhancer surfactants and chelators. Suitable surfactants include fatty acids and/or esters or salts thereof, bile acids and/or salts thereof. Suitable bile acids/salts include chenodeoxycholic acid (CDCA) and ursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitable fatty acids include arachidonic acid, undecanoic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a monoglyceride, a diglyceride or a pharmaceutically acceptable salt thereof (e.g., sodium). In some embodiments, combinations of penetration enhancers are used, for example, fatty acids/salts in combination with bile acids/salts. One exemplary combination is the sodium salt of lauric acid, capric acid and UDCA. Further penetration enhancers include polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAs featured in the invention can be delivered orally, in granular form including sprayed dried particles, or complexed to form micro or nanoparticles. DsRNA complexing agents include poly-amino acids; polyimines; polyacrylates; polyalkylacrylates, polyoxethanes, polyalkylcyanoacrylates; cationized gelatins, albumins, starches, acrylates, polyethyleneglycols (PEG) and starches; polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans, celluloses and starches. Suitable complexing agents include chitosan, N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine, polyspermines, protamine, polyvinylpyridine, polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g., p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate), poly(butylcyanoacrylate), poly(isobutylcyanoacrylate), poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate, DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate, polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolic acid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulations for dsRNAs and their preparation are described in detail in U.S. Pat. No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014, each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into the brain), intrathecal, intraventricular or intrahepatic administration can include sterile aqueous solutions which can also contain buffers, diluents and other suitable additives such as, but not limited to, penetration enhancers, carrier compounds and other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but are not limited to, solutions, emulsions, and liposome-containing formulations. These compositions can be generated from a variety of components that include, but are not limited to, preformed liquids, self-emulsifying solids and self-emulsifying semisolids. Particularly preferred are formulations that target the liver when treating hepatic disorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which can conveniently be presented in unit dosage form, can be prepared according to conventional techniques well known in the pharmaceutical industry. Such techniques include the step of bringing into association the active ingredients with the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the active ingredients with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

The compositions of the present invention can be formulated into any of many possible dosage forms such as, but not limited to, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories, and enemas. The compositions of the present invention can also be formulated as suspensions in aqueous, non-aqueous or mixed media. Aqueous suspensions can further contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

C. Additional Formulations

i. Emulsions

The compositions of the present invention can be prepared and formulated as emulsions. Emulsions are typically heterogeneous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions can be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions can contain additional components in addition to the dispersed phases, and the active drug which can be present as a solution in either aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants can also be present in emulsions as needed. Pharmaceutical emulsions can also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion can be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that can be incorporated into either phase of the emulsion. Emulsifiers can broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants can be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that can readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used can be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present invention, the compositions of iRNAs and nucleic acids are formulated as microemulsions. A microemulsion can be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) type is dependent on the properties of the oil and surfactant used and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott, in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1-propanol, and 1-butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions can, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase can typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase can include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri-glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (see e.g., U.S. Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci., 1996, 85, 138-143). Often microemulsions can form spontaneously when their components are brought together at ambient temperature. This can be particularly advantageous when formulating thermolabile drugs, peptides or iRNAs. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of iRNAs and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of iRNAs and nucleic acids.

Microemulsions of the present invention can also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the iRNAs and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention can be classified as belonging to one of five broad categories—surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these classes has been discussed above.

iii. Microparticles

an RNAi agent of the invention may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques.

iv. Penetration Enhancers

In one embodiment, the present invention employs various penetration enhancers to effect the efficient delivery of nucleic acids, particularly iRNAs, to the skin of animals. Most drugs are present in solution in both ionized and nonionized forms. However, usually only lipid soluble or lipophilic drugs readily cross cell membranes. It has been discovered that even non-lipophilic drugs can cross cell membranes if the membrane to be crossed is treated with a penetration enhancer. In addition to aiding the diffusion of non-lipophilic drugs across cell membranes, penetration enhancers also enhance the permeability of lipophilic drugs.

Penetration enhancers can be classified as belonging to one of five broad categories, i.e., surfactants, fatty acids, bile salts, chelating agents, and non-chelating non-surfactants (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Each of the above mentioned classes of penetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which, when dissolved in an aqueous solution, reduce the surface tension of the solution or the interfacial tension between the aqueous solution and another liquid, with the result that absorption of iRNAs through the mucosa is enhanced. In addition to bile salts and fatty acids, these penetration enhancers include, for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemical emulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Various fatty acids and their derivatives which act as penetration enhancers include, for example, oleic acid, lauric acid, capric acid (n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid, glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl and t-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate, caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g., Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol., 1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersion and absorption of lipids and fat-soluble vitamins (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's The Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts, and their synthetic derivatives, act as penetration enhancers. Thus the term “bile salts” includes any of the naturally occurring components of bile as well as any of their synthetic derivatives. Suitable bile salts include, for example, cholic acid (or its pharmaceutically acceptable sodium salt, sodium cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid (sodium glucholate), glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid (sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodium glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g., Malmsten, M. Surfactants and polymers in drug delivery, Informa Health Care, New York, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present invention, can be defined as compounds that remove metallic ions from solution by forming complexes therewith, with the result that absorption of iRNAs through the mucosa is enhanced. With regards to their use as penetration enhancers in the present invention, chelating agents have the added advantage of also serving as DNase inhibitors, as most characterized DNA nucleases require a divalent metal ion for catalysis and are thus inhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339). Suitable chelating agents include but are not limited to disodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates (e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acyl derivatives of collagen, laureth-9 and N-amino acyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. et al., Excipient development for pharmaceutical, biotechnology, and drug delivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancing compounds can be defined as compounds that demonstrate insignificant activity as chelating agents or as surfactants but that nonetheless enhance absorption of iRNAs through the alimentary mucosa (see e.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This class of penetration enhancers includes, for example, unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page 92); and non-steroidal anti-inflammatory agents such as diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level can also be added to the pharmaceutical and other compositions of the present invention. For example, cationic lipids, such as lipofectin (Junichi et al, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, and polycationic molecules, such as polylysine (Lollo et al., PCT Application WO 97/30731), are also known to enhance the cellular uptake of dsRNAs. Examples of commercially available transfection reagents include, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.), Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™ (Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad, Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX (Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen; Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.), RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen; Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENE Q2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAP Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPER Liposomal Transfection Reagent (Grenzacherstrasse, Switzerland), or Fugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega; Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison, Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent (Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille, France), EcoTransfect (OZ Biosciences; Marseille, France), TransPassa D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA), LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectin Transfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTER Transfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2 Transfection reagent (Genlantis; San Diego, Calif., USA), Cytofectin Transfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTER Transfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™ transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect (Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA), UniFECTOR (B-Bridge International; Mountain View, Calif., USA), SureFECTOR (B-Bridge International; Mountain View, Calif., USA), or HiFect™ (B-Bridge International, Mountain View, Calif., USA), among others.

Other agents can be utilized to enhance the penetration of the administered nucleic acids, including glycols such as ethylene glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenes such as limonene and menthone.

v. Carriers

Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, “carrier compound” or “carrier” can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor. For example, the recovery of a partially phosphorothioate dsRNA in hepatic tissue can be reduced when it is coadministered with polyinosinic acid, dextran sulfate, polycytidic acid or 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao et al., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl. Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or “excipient” is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient can be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can include sterile and non-sterile aqueous solutions, non-aqueous solutions in common solvents such as alcohols, or solutions of the nucleic acids in liquid or solid oil bases. The solutions can also contain buffers, diluents and other suitable additives. Pharmaceutically acceptable organic or inorganic excipients suitable for non-parenteral administration which do not deleteriously react with nucleic acids can be used.

Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

vii. Other Components

The compositions of the present invention can additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art-established usage levels. Thus, for example, the compositions can contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or can contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in the invention include (a) one or more iRNA compounds and (b) one or more agents which function by a non-RNAi mechanism and which are useful in treating a disorder of lipid metabolism.

Examples of such agents include, but are not limited to an anti-inflammatory agent, anti-steatosis agent, anti-viral, and/or anti-fibrosis agent. In addition, other substances commonly used to protect the liver, such as silymarin, can also be used in conjunction with the iRNAs described herein. Other agents useful for treating liver diseases include telbivudine, entecavir, and protease inhibitors such as telaprevir and other disclosed, for example, in Tung et al., U.S. Application Publication Nos. 2005/0148548, 2004/0167116, and 2003/0144217; and in Hale et al., U.S. Application Publication No. 2004/0127488.

Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD₅₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀. Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can be used in formulating a range of dosage for use in humans. The dosage of compositions featured herein in the invention lies generally within a range of circulating concentrations that include the ED₅₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the methods featured in the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range of the compound or, when appropriate, of the polypeptide product of a target sequence (e.g., achieving a decreased concentration of the polypeptide) that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

In addition to their administration, as discussed above, the iRNAs featured in the invention can be administered in combination with other known agents effective in treatment of pathological processes mediated by ANGPTL3 expression. In any event, the administering physician can adjust the amount and timing of iRNA administration on the basis of results observed using standard measures of efficacy known in the art or described herein.

VI. Methods of the Invention

The present invention also provides methods of using an iRNA of the invention and/or a composition containing an iRNA of the invention to reduce and/or inhibit ANGPTL3 expression in a cell. The methods include contacting the cell with a dsRNA of the invention and maintaining the cell for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known in the art. For example, a reduction in the expression of ANGPTL3 may be determined by determining the mRNA expression level of ANGPTL3 using methods routine to one of ordinary skill in the art, e.g., Northern blotting, qRT-PCR; by determining the protein level of ANGPTL3 using methods routine to one of ordinary skill in the art, such as Western blotting, immunological techniques. A reduction in the expression of ANGPTL3 may also be assessed indirectly by measuring a decrease in biological activity of ANGPTL3, e.g., a decrease in the level of serum lipid, triglycerides, cholesterol and/or free fatty acids.

In the methods of the invention the cell may be contacted in vitro or in vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the invention may be any cell that expresses an ANGPTL3gene. A cell suitable for use in the methods of the invention may be a mammalian cell, e.g., a primate cell (such as a human cell or a non-human primate cell, e.g., a monkey cell or a chimpanzee cell), a non-primate cell (such as a cow cell, a pig cell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbit cell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dog cell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell, or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), or a whale cell. In one embodiment, the cell is a human cell, e.g., a human liver cell.

ANGPTL3 expression is inhibited in the cell by at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or about 100%.

The in vivo methods of the invention may include administering to a subject a composition containing an iRNA, where the iRNA includes a nucleotide sequence that is complementary to at least a part of an RNA transcript of the ANGPTL3 gene of the mammal to be treated. When the organism to be treated is a mammal such as a human, the composition can be administered by any means known in the art including, but not limited to oral, intraperitoneal, or parenteral routes, including intracranial (e.g., intraventricular, intraparenchymal and intrathecal), intravenous, intramuscular, subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical (including buccal and sublingual) administration. In certain embodiments, the compositions are administered by intravenous infusion or injection. In certain embodiments, the compositions are administered by subcutaneous injection.

In some embodiments, the administration is via a depot injection. A depot injection may release the iRNA in a consistent way over a prolonged time period. Thus, a depot injection may reduce the frequency of dosing needed to obtain a desired effect, e.g., a desired inhibition of ANGPTL3, or a therapeutic or prophylactic effect. A depot injection may also provide more consistent serum concentrations. Depot injections may include subcutaneous injections or intramuscular injections. In preferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may be an external pump or a surgically implanted pump. In certain embodiments, the pump is a subcutaneously implanted osmotic pump. In other embodiments, the pump is an infusion pump. An infusion pump may be used for intravenous, subcutaneous, arterial, or epidural infusions. In preferred embodiments, the infusion pump is a subcutaneous infusion pump. In other embodiments, the pump is a surgically implanted pump that delivers the iRNA to the liver.

The mode of administration may be chosen based upon whether local or systemic treatment is desired and based upon the area to be treated. The route and site of administration may be chosen to enhance targeting.

In one aspect, the present invention also provides methods for inhibiting the expression of an ANGPTL3 gene in a mammal. The methods include administering to the mammal a composition comprising a dsRNA that targets an ANGPTL3 gene in a cell of the mammal and maintaining the mammal for a time sufficient to obtain degradation of the mRNA transcript of the ANGPTL3 gene, thereby inhibiting expression of the ANGPTL3 gene in the cell. Reduction in gene expression can be assessed by any methods known it the art and by methods, e.g. qRT-PCR, described herein. Reduction in protein production can be assessed by any methods known it the art and by methods, e.g. ELISA, described herein. In one embodiment, a puncture liver biopsy sample serves as the tissue material for monitoring the reduction in ANGPTL3 gene and/or protein expression.

The present invention further provides methods of treatment of a subject in need thereof. The treatment methods of the invention include administering an iRNA of the invention to a subject, e.g., a subject that would benefit from a reduction and/or inhibition of ANGPTL3 expression, in a therapeutically effective amount of an iRNA targeting an ANGPTL3 gene or a pharmaceutical composition comprising an iRNA targeting an ANGPTL3 gene.

An iRNA of the invention may be administered as a “free iRNA.” A free iRNA is administered in the absence of a pharmaceutical composition. The naked iRNA may be in a suitable buffer solution. The buffer solution may comprise acetate, citrate, prolamine, carbonate, or phosphate, or any combination thereof. In one embodiment, the buffer solution is phosphate buffered saline (PBS). The pH and osmolarity of the buffer solution containing the iRNA can be adjusted such that it is suitable for administering to a subject.

Alternatively, an iRNA of the invention may be administered as a pharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression are those having a disorder of lipid metabolism, e.g., an inherited disorder of lipid metabolism or an acquired disorder of lipid metabolism. In one embodiment, a subject having disorder of lipid metabolism has hyperlipidemia. In another embodiment, a subject having a disorder of lipid metabolism has hypertriglyceridemia. Treatment of a subject that would benefit from a reduction and/or inhibition of ANGPTL3 gene expression includes therapeutic treatment (e.g., a subject is having eruptive xanthomas) and prophylactic treatment (e.g., the subject is not having eruptive xanthomas or a subject may be at risk of developing eruptive xanthomas).

The invention further provides methods for the use of an iRNA or a pharmaceutical composition thereof, e.g., for treating a subject that would benefit from reduction and/or inhibition of ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, in combination with other pharmaceuticals and/or other therapeutic methods, e.g., with known pharmaceuticals and/or known therapeutic methods, such as, for example, those which are currently employed for treating these disorders. For example, in certain embodiments, an iRNA targeting ANGPTL3 is administered in combination with, e.g., an agent useful in treating a disorder of lipid metabolism as described elsewhere herein. For example, additional agents suitable for treating a subject that would benefit from reduction in ANGPTL3 expression, e.g., a subject having a disorder of lipid metabolism, may include agents that lower one or more serum lipids. Non-limiting examples of such agents may include cholesterol synthesis inhibitors, such as HMG-CoA reductase inhibitors, e.g., statins. Statins may include atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor), lovastatin extended-release (Altoprev), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor), and simvastatin (Zocor). Other agents useful in treating a disorder of lipid metabolism may include bile sequestering agents, such as cholestyramine and other resins; VLDL secretion inhibitors, such as niacin; lipophilic antioxidants, such as Probucol; acyl-CoA cholesterol acyl transferase inhibitors; farnesoid X receptor antagonists; sterol regulatory binding protein cleavage activating protein (SCAP) activators; microsomal triglyceride transfer protein (MTP) inhibitors; ApoE-related peptide; and therapeutic antibodies against ANGPTL3. The additional therapeutic agents may also include agents that raise high density lipoprotein (HDL), such as cholesteryl ester transfer protein (CETP) inhibitors. Furthermore, the additional therapeutic agents may also include dietary supplements, e.g., fish oil. The iRNA and additional therapeutic agents may be administered at the same time and/or in the same combination, e.g., parenterally, or the additional therapeutic agent can be administered as part of a separate composition or at separate times and/or by another method known in the art or described herein.

In one embodiment, the method includes administering a composition featured herein such that expression of the target ANGPTL3 gene is decreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24 hours, 28, 32, or about 36 hours. In one embodiment, expression of the target ANGPTL3 gene is decreased for an extended duration, e.g., at least about two, three, four days or more, e.g., about one week, two weeks, three weeks, or four weeks or longer.

Preferably, the iRNAs useful for the methods and compositions featured herein specifically target RNAs (primary or processed) of the target ANGPTL3gene. Compositions and methods for inhibiting the expression of these genes using iRNAs can be prepared and performed as described herein.

Administration of the dsRNA according to the methods of the invention may result in a reduction of the severity, signs, symptoms, and/or markers of such diseases or disorders in a patient with a disorder of lipid metabolism. By “reduction” in this context is meant a statistically significant decrease in such level. The reduction can be, for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, for example by measuring disease progression, disease remission, symptom severity, reduction in pain, quality of life, dose of a medication required to sustain a treatment effect, level of a disease marker or any other measurable parameter appropriate for a given disease being treated or targeted for prevention. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. For example, efficacy of treatment of a disorder of lipid metabolism may be assessed, for example, by periodic monitoring of one or more serum lipid levels. Comparisons of the later readings with the initial readings provide a physician an indication of whether the treatment is effective. It is well within the ability of one skilled in the art to monitor efficacy of treatment or prevention by measuring any one of such parameters, or any combination of parameters. In connection with the administration of an iRNA targeting ANGPTL3 or pharmaceutical composition thereof, “effective against” a disorder of lipid metabolism indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as a improvement of symptoms, a cure, a reduction in disease, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating disorder of lipid metabolisms and the related causes.

A treatment or preventive effect is evident when there is a statistically significant improvement in one or more parameters of disease status, or by a failure to worsen or to develop symptoms where they would otherwise be anticipated. As an example, a favorable change of at least 10% in a measurable parameter of disease, and preferably at least 20%, 30%, 40%, 50% or more can be indicative of effective treatment. Efficacy for a given iRNA drug or formulation of that drug can also be judged using an experimental animal model for the given disease as known in the art. When using an experimental animal model, efficacy of treatment is evidenced when a statistically significant reduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in the severity of disease as determined by one skilled in the art of diagnosis based on a clinically accepted disease severity grading scale, as but one example the Child-Pugh score (sometimes the Child-Turcotte-Pugh score). Any positive change resulting in e.g., lessening of severity of disease measured using the appropriate scale, represents adequate treatment using an iRNA or iRNA formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such as about 0.01 mg/kg to about 5 mg/kg, about 0.01 mg/kg to about 10 mg/kg, about 0.05 mg/kg to about 5 mg/kg, about 0.05 mg/kg to about 10 mg/kg, about 0.1 mg/kg to about 5 mg/kg, about 0.1 mg/kg to about 10 mg/kg, about 0.2 mg/kg to about 5 mg/kg, about 0.2 mg/kg to about 10 mg/kg, about 0.3 mg/kg to about 5 mg/kg, about 0.3 mg/kg to about 10 mg/kg, about 0.4 mg/kg to about 5 mg/kg, about 0.4 mg/kg to about 10 mg/kg, about 0.5 mg/kg to about 5 mg/kg, about 0.5 mg/kg to about 10 mg/kg, about 1 mg/kg to about 5 mg/kg, about 1 mg/kg to about 10 mg/kg, about 1.5 mg/kg to about 5 mg/kg, about 1.5 mg/kg to about 10 mg/kg, about 2 mg/kg to about about 2.5 mg/kg, about 2 mg/kg to about 10 mg/kg, about 3 mg/kg to about 5 mg/kg, about 3 mg/kg to about 10 mg/kg, about 3.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 5 mg/kg, about 4.5 mg/kg to about 5 mg/kg, about 4 mg/kg to about 10 mg/kg, about 4.5 mg/kg to about 10 mg/kg, about 5 mg/kg to about 10 mg/kg, about 5.5 mg/kg to about 10 mg/kg, about 6 mg/kg to about 10 mg/kg, about 6.5 mg/kg to about 10 mg/kg, about 7 mg/kg to about 10 mg/kg, about 7.5 mg/kg to about 10 mg/kg, about 8 mg/kg to about 10 mg/kg, about 8.5 mg/kg to about 10 mg/kg, about 9 mg/kg to about 10 mg/kg, or about 9.5 mg/kg to about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, the dsRNA may be administered at a dose of about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, or about 10 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

In other embodiments, for example, when a composition of the invention comprises a dsRNA as described herein and an N-acetylgalactosamine, subjects can be administered a therapeutic amount of dsRNA, such as a dose of about 0.1 to about 50 mg/kg, about 0.25 to about 50 mg/kg, about 0.5 to about 50 mg/kg, about 0.75 to about 50 mg/kg, about 1 to about 50 mg/mg, about 1.5 to about 50 mg/kb, about 2 to about 50 mg/kg, about 2.5 to about 50 mg/kg, about 3 to about 50 mg/kg, about 3.5 to about 50 mg/kg, about 4 to about 50 mg/kg, about 4.5 to about 50 mg/kg, about 5 to about 50 mg/kg, about 7.5 to about 50 mg/kg, about 10 to about 50 mg/kg, about 15 to about 50 mg/kg, about 20 to about 50 mg/kg, about 20 to about 50 mg/kg, about 25 to about 50 mg/kg, about 25 to about 50 mg/kg, about 30 to about 50 mg/kg, about 35 to about 50 mg/kg, about 40 to about 50 mg/kg, about 45 to about 50 mg/kg, about 0.1 to about 45 mg/kg, about 0.25 to about 45 mg/kg, about 0.5 to about 45 mg/kg, about 0.75 to about 45 mg/kg, about 1 to about 45 mg/mg, about 1.5 to about 45 mg/kb, about 2 to about 45 mg/kg, about 2.5 to about 45 mg/kg, about 3 to about 45 mg/kg, about 3.5 to about 45 mg/kg, about 4 to about 45 mg/kg, about 4.5 to about 45 mg/kg, about 5 to about 45 mg/kg, about 7.5 to about 45 mg/kg, about 10 to about 45 mg/kg, about 15 to about 45 mg/kg, about 20 to about 45 mg/kg, about 20 to about 45 mg/kg, about 25 to about 45 mg/kg, about 25 to about 45 mg/kg, about 30 to about 45 mg/kg, about 35 to about 45 mg/kg, about 40 to about 45 mg/kg, about 0.1 to about 40 mg/kg, about 0.25 to about 40 mg/kg, about 0.5 to about 40 mg/kg, about 0.75 to about 40 mg/kg, about 1 to about 40 mg/mg, about 1.5 to about 40 mg/kb, about 2 to about 40 mg/kg, about 2.5 to about 40 mg/kg, about 3 to about 40 mg/kg, about 3.5 to about 40 mg/kg, about 4 to about 40 mg/kg, about 4.5 to about 40 mg/kg, about 5 to about 40 mg/kg, about 7.5 to about 40 mg/kg, about 10 to about 40 mg/kg, about 15 to about 40 mg/kg, about 20 to about 40 mg/kg, about 20 to about 40 mg/kg, about 25 to about 40 mg/kg, about 25 to about 40 mg/kg, about 30 to about 40 mg/kg, about 35 to about 40 mg/kg, about 0.1 to about 30 mg/kg, about 0.25 to about 30 mg/kg, about 0.5 to about 30 mg/kg, about 0.75 to about 30 mg/kg, about 1 to about 30 mg/mg, about 1.5 to about 30 mg/kb, about 2 to about 30 mg/kg, about 2.5 to about 30 mg/kg, about 3 to about 30 mg/kg, about 3.5 to about 30 mg/kg, about 4 to about 30 mg/kg, about 4.5 to about 30 mg/kg, about 5 to about 30 mg/kg, about 7.5 to about 30 mg/kg, about 10 to about 30 mg/kg, about 15 to about 30 mg/kg, about 20 to about 30 mg/kg, about 20 to about 30 mg/kg, about 25 to about 30 mg/kg, about 0.1 to about 20 mg/kg, about 0.25 to about 20 mg/kg, about 0.5 to about 20 mg/kg, about 0.75 to about 20 mg/kg, about 1 to about 20 mg/mg, about 1.5 to about 20 mg/kb, about 2 to about 20 mg/kg, about 2.5 to about 20 mg/kg, about 3 to about 20 mg/kg, about 3.5 to about 20 mg/kg, about 4 to about 20 mg/kg, about 4.5 to about 20 mg/kg, about 5 to about 20 mg/kg, about 7.5 to about 20 mg/kg, about 10 to about 20 mg/kg, or about 15 to about 20 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

For example, subjects can be administered a therapeutic amount of dsRNA, such as about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8. 1.9, 2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8. 2.9, 3, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8. 3.9, 4, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8. 4.9, 5, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8. 5.9, 6, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8. 6.9, 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8. 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8. 8.9, 9, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8. 9.9, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mg/kg. Values and ranges intermediate to the recited values are also intended to be part of this invention.

The iRNA can be administered by intravenous infusion over a period of time, such as over a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or about a 25 minute period. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. For example, after administration biweekly for three months, administration can be repeated once per month, for six months or a year or longer. Administration of the iRNA can reduce ANGPTL3 levels, e.g., in a cell, tissue, blood, urine or other compartment of the patient by at least about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or at least about 99% or more.

Before administration of a full dose of the iRNA, patients can be administered a smaller dose, such as a 5% infusion reaction, and monitored for adverse effects, such as an allergic reaction. In another example, the patient can be monitored for unwanted immunostimulatory effects, such as increased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Alternatively, the iRNA can be administered subcutaneously, i.e., by subcutaneous injection. One or more injections may be used to deliver the desired daily dose of iRNA to a subject. The injections may be repeated over a period of time, such as over 2, 3, 4, 5, 6, 7, 8, 9, 10 or 15 days. The administration may be repeated, for example, on a regular basis, such as biweekly (i.e., every two weeks) for one month, two months, three months, four months or longer. After an initial treatment regimen, the treatments can be administered on a less frequent basis. In some embodiments, a single dose of iRNA is followed by monthly dosing. In some embodiments, the dosing may comprise a loading phase of multiple doses on consecutive days.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the iRNAs and methods featured in the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

EXAMPLES Example 1. iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, such reagent can be obtained from any supplier of reagents for molecular biology at a quality/purity standard for application in molecular biology.

Transcripts

siRNA design was carried out to identify siRNAs targeting the human ANGPTL3 transcript annotated in the NCBI Gene database (http://www.ncbi.nlm.nih.gov/gene/) and a cynomolgus monkey (Macaca fascicularis; henceforth “cyno”) ANGPTL3 transcript produced via sequencing of cDNA prepared from liver RNA. Sequencing of cyno ANGPTL3 mRNA was done in-house, and the mRNA sequence is shown in SEQ ID NO:9. Design used the following transcripts from the NCBI collection: Human—NM_014495.2 (SEQ ID NO:1); Mouse—NM_013913.3 (SEQ ID NO:2). All siRNA duplexes were designed that shared 100% identity with the listed human and cyno transcripts. A subset of siRNA duplexes, described below, also shared 100% identity with the mouse (Mus musculus) ANGPTL3 transcript found in NCBI Gene database.

siRNA Design, Specificity, and Efficacy Prediction

The predicted specificity of all possible 19mers was predicted from each sequence. Candidate 19mers were then selected that lacked repeats longer than 7 nucleotides. These 977 candidate human/cyno siRNAs, and a subset of 38 that also matched mouse (“human/cyno/mouse candidate siRNAs”) were then used in a comprehensive search against the human transcriptome (defined as the set of NM_and XM_records within the human NCBI Refseq set) using an exhaustive “brute-force” algorithm implemented in the python script ‘BruteForce.py’. The script next parsed the transcript-oligo alignments to generate a score based on the position and number of mismatches between the siRNA and any potential ‘off-target’ transcript. The off-target score is weighted to emphasize differences in the ‘seed’ region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Each oligo-transcript pair from the brute-force search was given a mismatch score by summing the individual mismatch scores; mismatches in the position 2-9 were counted as 2.8, mismatches in the cleavage site positions 10-11 were counted as 1.2, and mismatches in region 12-19 counted as 1.0. An additional off-target prediction was carried out by comparing the frequency of heptamers and octomers derived from 3 distinct, seed-derived hexamers of each oligo. The hexamers from positions 2-7 relative to the 5′ start were used to create 2 heptamers and one octomer. ‘Heptamer1’ was created by adding a 3′ A to the hexamer; ‘heptamer2’ was created by adding a 5′ A to the hexamer; octomer was created by adding an A to both 5′ and 3′ ends of the hexamer. The frequency of octomers and heptamers in the human 3′UTRome (defined as the subsequence of the transcriptome from NCBI's Refseq database where the end of the coding region, the ‘CDS’, is clearly defined) was pre-calculated. The octomer frequency was normalized to the heptamer frequency using the median value from the range of octomer frequencies. A ‘mirSeedScore’ was then calculated by calculating the sum of ((3× normalized octomer count)+(2× heptamer2 count)+(1× heptamer1 count)).

Both siRNAs strands were assigned to a category of specificity according to the calculated scores: a score above 3 qualifies as highly specific, equal to 3 as specific and between 2.2 and 2.8 as moderately specific. Sorting was carried out by the specificity of the antisense strand. Duplexes were then selected from the human/cyno set with antisense oligos lacking miRNA seed matches, scores of 3 or better, less than 65% overall GC content, no GC at the first position, 4 or more Us or As in the seed region, and GC at the nineteenth position. Duplexes from the human/cyno/mouse set with antisense oligos having scores of 2 or better, less than 65% overall GC content, and no GC at the first position were also selected.

siRNA Sequence Selection

A total of 47 sense and 47 antisense derived siRNA oligos from the human/cyno set were synthesized and formed into duplexes. A total of 15 sense and 15 antisense derived siRNAs from the human/cyno/mouse set were synthesized and formed into duplexes.

Synthesis of ANGPTL3 Sequences

ANGPTL3 sequences were synthesized on a MerMade 192 synthesizer at either a 1 or 0.2 μmol scale. Single strands were synthesized with 2′O-methyl modifications for transfection based in vitro screening. For use in free uptake screening assays, 3′ GalNAc conjugates were made with 2′F and 2′-O-methyl chemical modifications. In these designs, GalNAc moiety was placed at the 3′end of the sense strand. The antisense sequence was 23 nucleotides in length and also contained 2′F and 2′Omethyl chemical modifications with two phosphorothioate linkages at the 3′end.

On one set of 21mer single strands and duplexes, ‘endolight’ chemistry was applied as detailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand were         modified with 2′-O-Methyl nucleotides (2′ O-Methyl C and         2′-O-Methyl U)     -   In the antisense strand, pyrimidines adjacent (towards 5′         position) to ribo A nucleoside were replaced with their         corresponding 2′-O-Methyl nucleosides     -   A two base dTsdT extension at the 3′ end of both sense and anti         sense sequences was introduced

For GalNAc conjugated 21mer sense and complementary 23mer antisense sequences, 2′F and 2′OMethyl modified single strands were synthesized. The synthesis was performed on a GalNAc modified CPG support for the sense strand and CPG modified with universal support for the antisense sequence at a 1 μmol scale. The sequence motif named TOFFEE was applied, in which the sense strand contained a three-nucleotide 2′F-modified motif at positions 9, 10 and 11 and in the antisense, a 2′OMethyl-modified motif was included at positions 11, 12 and 13.

Synthesis, Cleavage and Deprotection

The synthesis of ANGPTL3 sequences used solid supported oligonucleotide synthesis using phosphoramidite chemistry. For 21 mer endolight sequences, a deoxy thymidine CPG was used as the solid support while for the GalNAc conjugates, GalNAc solid support for the sense strand and a universal CPG for the antisesense strand were used.

The synthesis of the above sequences was performed at either a 1 or 0.2 μm scale in 96 well plates. The amidite solutions were prepared at 0.1M concentration and ethyl thio tetrazole (0.6M in Acetonitrile) was used as the activator.

The synthesized sequences were cleaved and deprotected in 96 well plates, using methylamine in the first step and fluoride reagent in the second step. For GalNAc and 2′F nucleoside containing sequences, deprotection conditions were modified. Sequences after cleavage and deprotection were precipitated using an acetone:ethanol (80:20) mix and the pellets were re-suspended in 0.2M sodium acetate buffer. Samples from each sequence were analyzed by LC-MS to confirm the identity, UV for quantification and a selected set of samples by IEX chromatography to determine purity.

Purification, Desalting and Annealing

ANGPTL3 sequences were precipitated and purified on an AKTA Purifier system using a Sephadex column. The ANGPTL3 was run at ambient temperature. Sample injection and collection was performed in 96 well plates with 1.8 mL deep wells. A single peak corresponding to the full length sequence was collected in the eluent. The desalted ANGPTL3 sequences were analyzed for concentration (by UV measurement at A₂₆₀) and purity (by ion exchange HPLC). The complementary single strands were then combined in a 1:1 stoichiometric ratio to form siRNA duplexes.

Example 2. In Vitro Screening

Cell Culture and Transfections

Hep3B cells (ATCC, Manassas, Va.) were grown to near confluence at 37° C. in an atmosphere of 5% CO₂ in RPMI (ATCC) supplemented with 10% FBS, streptomycin, and glutamine (ATCC) before being released from the plate by trypsinization. Transfection was carried out by adding 14.8 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNA duplexes per well into a 96-well plate and incubated at room temperature for 15 minutes. 80 μl of complete growth media without antibiotic containing ˜2×10⁴ Hep3B cells were then added to the siRNA mixture. Cells were incubated for either 24 or 120 hours prior to RNA purification. Single dose experiments were performed at 10 nM and 0.1 nM final duplex concentration and dose response experiments were done at 10, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 and 0.00001 nM final duplex concentration unless otherwise stated.

Free Uptake Transfection

5 μl of each GalNac conjugated siRNA in PBS was combined with 4×10⁴ freshly thawed cryopreserved Cynomolgus monkey hepatocytes resuspended in 95 μl of In Vitro Gro CP media (In Vitro Technologies—Celsis, Baltimore, Md.) in each well of a 96 well plate. The mixture was incubated for about 24 hrs at 37° C. in an atmosphere of 5% CO₂. siRNAs were tested at final concentrations of 500 nM, 100 nM and 10 nM for efficacy free uptake assays. For dose response screens, final siRNA concentrations were 500 nM, 100 nM, 20 nM, 4 nM, 0.8 nM, 0.16 nM, 0.032 nM and 0.0064 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part #: 610-12)

Cells were harvested and lysed in 150 μl of Lysis/Binding Buffer then mixed for 5 minute at 850 rpm using an Eppendorf Thermomixer (the mixing speed was the same throughout the process). Ten microliters of magnetic beads and 80 μl of Lysis/Binding Buffer mixture were added to a round bottom plate and mixed for 1 minute. Magnetic beads were captured using magnetic stand and the supernatant was removed without disturbing the beads. After removing supernatant, the lysed cells were added to the remaining beads and mixed for 5 minutes. After removing supernatant, magnetic beads were washed 2 times with 150 μl Wash Buffer A and mixed for 1 minute. Beads were captured again and supernatant removed. Beads were then washed with 150 μl of Wash Buffer B, captured, and the supernatant was removed. Beads were next washed with 150 μl Elution Buffer, captured, and the supernatant was removed. Beads were allowed to dry for 2 minutes. After drying, 50 μl of Elution Buffer was added and mixed for 5 minutes at 70° C. Beads were captured on magnet for 5 minutes. 40 μl of supernatant was removed and added to another 96 well plate.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, Calif., Cat #4368813)

A master mix of 2 μl 10× Buffer, 0.8 μl 25×dNTPs, 2 μl Random primers, 1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H₂O per reaction were added into 10 μl total RNA. cDNA was generated using a Bio-Rad C-1000 or S—1000 thermal cycler (Hercules, Calif.) through the following steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C. hold.

Real Time PCR

2 μl of cDNA was added to a master mix containing 0.5 μl GAPDH TaqMan Probe (Applied Biosystems Cat #4326317E), 0.5 μl ANGPTL TaqMan probe (Applied Biosystems cat # Hs00205581_m1) and 50 Lightcycler 480 probe master mix (Roche Cat #04887301001) per well in a 384 well 50 plates (Roche cat #04887301001). Real time PCR was done in an ABI 7900HT Real Time PCR system (Applied Biosystems) using the ΔΔCt(RQ) assay. Each duplex was tested in two independent transfections, and each transfection was assayed in duplicate, unless otherwise noted in the summary tables.

To calculate relative fold change, real time data was analyzed using the ΔΔCt method and normalized to assays performed with cells transfected with 10 nM AD-1955, or mock transfected cells. IC₅₀s were calculated using a 4 parameter fit model using XLFit and normalized to cells transfected with AD-1955 or naïve cells over the same dose range, or to its own lowest dose. AD-1955 sequence, used as a negative control, targets luciferase and has the following sequence: sense: cuuAcGcuGAGuAcuucGAdTsdT (SEQ ID NO: 14); antisense: UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO: 15).

Viability Screens

Cell viability was measured on days 3 and 6 in HeLa and Hep3B cells following transfection with 10, 1, 0.5, 0.1, 0.05 nM siRNA. Cells were plated at a density of 10,000 cells per well in 96 well plates. Each siRNA was assayed in triplicate and the data averaged. siRNAs targeting PLK1 and AD-19200 were included as positive controls for loss of viability, and AD-1955 and mock transfected cells as negative controls. PLK1 and AD-19200 result in a dose dependent loss of viability. To measure viability, 20 μl of CellTiter Blue (Promega) was added to each well of the 96 well plates after 3 or 6 days and incubated at 37° C. for 2 hours. Plates were then read in a Spectrophotometer (Molecular Devices) at 560Ex/590Em. Viability was expressed as the average value of light units from three replicate transfections+/−standard deviation. Relative viability was assessed by first averaging the three replicate transfections and then normalizing Mock transfected cells. Data is expressed as % viable cells.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acid sequence representation. It will be understood that these monomers, when present in an oligonucleotide, are mutually linked by 5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A adenosine C cytidine G guanosine T thymidine U uridine N any nucleotide (G, A, C, T or U) a 2′-O-methyladenosine c 2′-O-methylcytidine g 2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine s phosphorothioate linkage

TABLE 2 Unmodified sense and antisense strand sequences of ANGPTL3 dsRNAs Sense Sequence Antisense Sequence (SEQ ID NOS 16-77, (SEQ ID NOS 78-139, Sense respectively, in Position in Antisense respectively, in Position in Duplex ID Name order of appearance) NM_014495.2 Name order of appearance) NM_014495.2 AD-45939.1 A-96225.1 UAUUUGAUCAGUCUUUUUA 281-299 A-96226.1 UAAAAAGACUGAUCAAAUA 281-299 AD-45858.1 A-96149.1 GAGCAACUAACUAACUUAA 478-496 A-96150.1 UUAAGUUAGUUAGUUGCUC 478-496 AD-45869.1 A-96137.1 GGCCAAAUUAAUGACAUAU 247-265 A-96138.1 AUAUGUCAUUAAUUUGGCC 247-265 AD-45884.1 A-96189.1 CGAAUUGAGUUGGAAGACU 1045-1063 A-96190.1 AGUCUUCCAACUCAAUUCG 1045-1063 AD-45892.1 A-96129.1 CCUCCUUCAGUUGGGACAU 198-216 A-96130.1 AUGUCCCAACUGAAGGAGG 198-216 AD-45899.1 A-96147.1 CACUUGAACUCAACUCAAA 401-419 A-96148.1 UUUGAGUUGAGUUCAAGUG 401-419 AD-45915.1 A-96231.1 GUCCAUGGACAUUAAUUCA 890-908 A-96232.1 UGAAUUAAUGUCCAUGGAC 890-908 AD-45924.1 A-96219.1 AAUCAAGAUUUGCUAUGUU 152-170 A-96220.1 AACAUAGCAAAUCUUGAUU 152-170 AD-45860.1 A-96181.1 CUAGAGAAGAUAUACUCCA 1000-1018 A-96182.1 UGGAGUAUAUCUUCUCUAG 1000-1018 AD-45870.1 A-96153.1 CUAACUAACUUAAUUCAAA 484-502 A-96154.1 UUUGAAUUAAGUUAGUUAG 484-502 AD-45870.2 A-96153.2 CUAACUAACUUAAUUCAAA 484-502 A-96154.2 UUUGAAUUAAGUUAGUUAG 484-502 AD-45877.1 A-96171.1 CAUUAAUUCAACAUCGAAU 899-917 A-96172.1 AUUCGAUGUUGAAUUAAUG 899-917 AD-45885.1 A-96205.1 CAAAAUGUUGAUCCAUCCA 1392-1410 A-96206.1 UGGAUGGAUCAACAUUUUG 1392-1410 AD-45893.1 A-96145.1 CAUAUAAACUACAAGUCAA 359-377 A-96146.1 UUGACUUGUAGUUUAUAUG 359-377 AD-45900.1 A-96163.1 GACCCAGCAACUCUCAAGU 839-857 A-96164.1 ACUUGAGAGUUGCUGGGUC 839-857 AD-45925.1 A-96235.1 GGUUGGGCCUAGAGAAGAU  992-1010 A-96236.1 AUCUUCUCUAGGCCCAACC  992-1010 AD-45861.1 A-96197.1 GUGUGGAGAAAACAACCUA 1272-1290 A-96198.1 UAGGUUGUUUUCUCCACAC 1272-1290 AD-45871.1 A-96169.1 GACAUUAAUUCAACAUCGA 897-915 A-96170.1 UCGAUGUUGAAUUAAUGUC 897-915 AD-45878.1 A-96187.1 CAUAGUGAAGCAAUCUAAU 1017-1035 A-96188.1 AUUAGAUUGCUUCACUAUG 1017-1035 AD-45886.1 A-96127.1 CUAUGUUAGACGAUGUAAA 164-182 A-96128.1 UUUACAUCGUCUAACAUAG 164-182 AD-45894.1 A-96161.1 CACAGAAAUUUCUCUAUCU 684-702 A-96162.1 AGAUAGAGAAAUUUCUGUG 684-702 AD-45901.1 A-96179.1 GUUGGGCCUAGAGAAGAUA  993-1011 A-96180.1 UAUCUUCUCUAGGCCCAAC  993-1011 AD-45909.1 A-96213.1 GCCAAAAUCAAGAUUUGCU 147-165 A-96214.1 AGCAAAUCUUGAUUUUGGC 147-165 AD-45934.1 A-96223.1 ACAUAUUUGAUCAGUCUUU 278-296 A-96224.1 AAAGACUGAUCAAAUAUGU 278-296 AD-45934.2 A-96223.2 ACAUAUUUGAUCAGUCUUU 278-296 A-96224.2 AAAGACUGAUCAAAUAUGU 278-296 AD-45863.1 A-96135.1 CUUAAAGACUUUGUCCAUA 220-238 A-96136.1 UAUGGACAAAGUCUUUAAG 220-238 AD-45872.1 A-96185.1 CCAUAGUGAAGCAAUCUAA 1016-1034 A-96186.1 UUAGAUUGCUUCACUAUGG 1016-1034 AD-45879.1 A-96203.1 CAACCAAAAUGUUGAUCCA 1388-1406 A-96204.1 UGGAUCAACAUUUUGGUUG 1388-1406 AD-45887.1 A-96143.1 CUACAUAUAAACUACAAGU 356-374 A-96144.1 ACUUGUAGUUUAUAUGUAG 356-374 AD-45895.1 A-96177.1 GGGAGGCUUGAUGGAGAAU 970-988 A-96178.1 AUUCUCCAUCAAGCCUCCC 970-988 AD-45902.1 A-96195.1 GGUGUUUUCUACUUGGGAU 1188-1206 A-96196.1 AUCCCAAGUAGAAAACACC 1188-1206 AD-45910.1 A-96229.1 AAGAGCACCAAGAACUACU 711-729 A-96230.1 AGUAGUUCUUGGUGCUCUU 711-729 AD-45935.1 A-96239.1 UGGAGAAAACAACCUAAAU 1275-1293 A-96240.1 AUUUAGGUUGUUUUCUCCA 1275-1293 AD-45864.1 A-96151.1 GCAACUAACUAACUUAAUU 480-498 A-96152.1 AAUUAAGUUAGUUAGUUGC 480-498 AD-45873.1 A-96201.1 CAACCUAAAUGGUAAAUAU 1284-1302 A-96202.1 AUAUUUACCAUUUAGGUUG 1284-1302 AD-45880.1 A-96125.1 GCUAUGUUAGACGAUGUAA 163-181 A-96126.1 UUACAUCGUCUAACAUAGC 163-181 AD-45888.1 A-96159.1 CCCACAGAAAUUUCUCUAU 682-700 A-96160.1 AUAGAGAAAUUUCUGUGGG 682-700 AD-45896.1 A-96193.1 GAUUUGGUGUUUUCUACUU 1183-1201 A-96194.1 AAGUAGAAAACACCAAAUC 1183-1201 AD-45903.1 A-96211.1 CAGAGCCAAAAUCAAGAUU 143-161 A-96212.1 AAUCUUGAUUUUGGCUCUG 143-161 AD-45919.1 A-96217.1 AAAUCAAGAUUUGCUAUGU 151-169 A-96218.1 ACAUAGCAAAUCUUGAUUU 151-169 AD-45865.1 A-96167.1 CAUGGACAUUAAUUCAACA 893-911 A-96168.1 UGUUGAAUUAAUGUCCAUG 893-911 AD-45874.1 A-96123.1 GAUUUGCUAUGUUAGACGA 158-176 A-96124.1 UCGUCUAACAUAGCAAAUC 158-176 AD-45881.1 A-96141.1 GAACUACAUAUAAACUACA 353-371 A-96142.1 UGUAGUUUAUAUGUAGUUC 353-371 AD-45889.1 A-96175.1 CGAAUAGAUGGAUCACAAA 913-931 A-96176.1 UUUGUGAUCCAUCUAUUCG 913-931 AD-45897.1 A-96209.1 CUUGUUAAAACUCUAAACU 1817-1835 A-96210.1 AGUUUAGAGUUUUAACAAG 1817-1835 AD-45904.1 A-96227.1 AUUUGAUCAGUCUUUUUAU 282-300 A-96228.1 AUAAAAAGACUGAUCAAAU 282-300 AD-45920.1 A-96233.1 UCCAUGGACAUUAAUUCAA 891-909 A-96234.1 UUGAAUUAAUGUCCAUGGA 891-909 AD-45856.1 A-96117.1 CACAAUUAAGCUCCUUCUU 57-75 A-96118.1 AAGAAGGAGCUUAAUUGUG 57-75 AD-45929.1 A-96221.1 CAACAUAUUUGAUCAGUCU 276-294 A-96222.1 AGACUGAUCAAAUAUGUUG 276-294 AD-45866.1 A-96183.1 CUCCAUAGUGAAGCAAUCU 1014-1032 A-96184.1 AGAUUGCUUCACUAUGGAG 1014-1032 AD-45875.1 A-96139.1 GCCAAAUUAAUGACAUAUU 248-266 A-96140.1 AAUAUGUCAUUAAUUUGGC 248-266 AD-45882.1 A-96157.1 CAACAGCAUAGUCAAAUAA 622-640 A-96158.1 UUAUUUGACUAUGCUGUUG 622-640 AD-45890.1 A-96191.1 GGAAAUCACGAAACCAACU 1105-1123 A-96192.1 AGUUGGUUUCGUGAUUUCC 1105-1123 AD-45898.1 A-96131.1 CAGUUGGGACAUGGUCUUA 205-223 A-96132.1 UAAGACCAUGUCCCAACUG 205-223 AD-45857.1 A-96133.1 GACAUGGUCUUAAAGACUU 212-230 A-96134.1 AAGUCUUUAAGACCAUGUC 212-230 AD-45930.1 A-96237.1 UGUGGAGAAAACAACCUAA 1273-1291 A-96238.1 UUAGGUUGUUUUCUCCACA 1273-1291 AD-45867.1 A-96199.1 GUGGAGAAAACAACCUAAA 1274-1292 A-96200.1 UUUAGGUUGUUUUCUCCAC 1274-1292 AD-45876.1 A-96155.1 CCAACAGCAUAGUCAAAUA 621-639 A-96156.1 UAUUUGACUAUGCUGUUGG 621-639 AD-45883.1 A-96173.1 CAACAUCGAAUAGAUGGAU 907-925 A-96174.1 AUCCAUCUAUUCGAUGUUG 907-925 AD-45891.1 A-96207.1 GCAAAUUUAAAAGGCAAUA 1441-1459 A-96208.1 UAUUGCCUUUUAAAUUUGC 1441-1459 AD-45914.1 A-96215.1 CAAAAUCAAGAUUUGCUAU 149-167 A-96216.1 AUAGCAAAUCUUGAUUUUG 149-167 AD-15838.1 A-26242.1 AGAGCCAAAAUCAAGAUUU 144-162 A-26243.2 AAAUCUUGAUUUUGGCUCU 144-162

TABLE 3 Modified sense and antisense strand sequences of ANGPTL3 dsRNAs Sense Sequence Antisense Sequence (SEQ ID NOS 140-201, (SEQ ID NOS 202-263, Duplex Sense respectively, in Antisense respectively, in ID OligoName order of appearance) OligoName order of appearance) AD-45939.1 A-96225.1 uAuuuGAucAGucuuuuuAdTsdT A-96226.1 uAAAAAGACUGAUcAAAuAdTsdT AD-45858.1 A-96149.1 GAGcAAcuAAcuAAcuuAAdTsdT A-96150.1 UuAAGUuAGUuAGUUGCUCdTsdT AD-45869.1 A-96137.1 GGccAAAuuAAuGAcAuAudTsdT A-96138.1 AuAUGUcAUuAAUUUGGCCdTsdT AD-45884.1 A-96189.1 cGAAuuGAGuuGGAAGAcudTsdT A-96190.1 AGUCUUCcAACUcAAUUCGdTsdT AD-45892.1 A-96129.1 ccuccuucAGuuGGGAcAudTsdT A-96130.1 AUGUCCcAACUGAAGGAGGdTsdT AD-45899.1 A-96147.1 cAcuuGAAcucAAcucAAAdTsdT A-96148.1 UUUGAGUUGAGUUcAAGUGdTsdT AD-45915.1 A-96231.1 GuccAuGGAcAuuAAuucAdTsdT A-96232.1 UGAAUuAAUGUCcAUGGACdTsdT AD-45924.1 A-96219.1 AAucAAGAuuuGcuAuGuudTsdT A-96220.1 AAcAuAGcAAAUCUUGAUUdTsdT AD-45860.1 A-96181.1 cuAGAGAAGAuAuAcuccAdTsdT A-96182.1 UGGAGuAuAUCUUCUCuAGdTsdT AD-45870.1 A-96153.1 cuAAcuAAcuuAAuucAAAdTsdT A-96154.1 UUUGAAUuAAGUuAGUuAGdTsdT AD-45870.2 A-96153.2 cuAAcuAAcuuAAuucAAAdTsdT A-96154.2 UUUGAAUuAAGUuAGUuAGdTsdT AD-45877.1 A-96171.1 cAuuAAuucAAcAucGAAudTsdT A-96172.1 AUUCGAUGUUGAAUuAAUGdTsdT AD-45885.1 A-96205.1 cAAAAuGuuGAuccAuccAdTsdT A-96206.1 UGGAUGGAUcAAcAUUUUGdTsdT AD-45893.1 A-96145.1 cAuAuAAAcuAcAAGucAAdTsdT A-96146.1 UUGACUUGuAGUUuAuAUGdTsdT AD-45900.1 A-96163.1 GAcccAGcAAcucucAAGudTsdT A-96164.1 ACUUGAGAGUUGCUGGGUCdTsdT AD-45925.1 A-96235.1 GGuuGGGccuAGAGAAGAudTsdT A-96236.1 AUCUUCUCuAGGCCcAACCdTsdT AD-45861.1 A-96197.1 GuGuGGAGAAAAcAAccuAdTsdT A-96198.1 uAGGUUGUUUUCUCcAcACdTsdT AD-45871.1 A-96169.1 GAcAuuAAuucAAcAucGAdTsdT A-96170.1 UCGAUGUUGAAUuAAUGUCdTsdT AD-45878.1 A-96187.1 cAuAGuGAAGcAAucuAAudTsdT A-96188.1 AUuAGAUUGCUUcACuAUGdTsdT AD-45886.1 A-96127.1 cuAuGuuAGAcGAuGuAAAdTsdT A-96128.1 UUuAcAUCGUCuAAcAuAGdTsdT AD-45894.1 A-96161.1 cAcAGAAAuuucucuAucudTsdT A-96162.1 AGAuAGAGAAAUUUCUGUGdTsdT AD-45901.1 A-96179.1 GuuGGGccuAGAGAAGAuAdTsdT A-96180.1 uAUCUUCUCuAGGCCcAACdTsdT AD-45909.1 A-96213.1 GccAAAAucAAGAuuuGcudTsdT A-96214.1 AGcAAAUCUUGAUUUUGGCdTsdT AD-45934.1 A-96223.1 AcAuAuuuGAucAGucuuudTsdT A-96224.1 AAAGACUGAUcAAAuAUGUdTsdT AD-45934.2 A-96223.2 AcAuAuuuGAucAGucuuudTsdT A-96224.2 AAAGACUGAUcAAAuAUGUdTsdT AD-45863.1 A-96135.1 cuuAAAGAcuuuGuccAuAdTsdT A-96136.1 uAUGGAcAAAGUCUUuAAGdTsdT AD-45872.1 A-96185.1 ccAuAGuGAAGcAAucuAAdTsdT A-96186.1 UuAGAUUGCUUcACuAUGGdTsdT AD-45879.1 A-96203.1 cAAccAAAAuGuuGAuccAdTsdT A-96204.1 UGGAUcAAcAUUUUGGUUGdTsdT AD-45887.1 A-96143.1 cuAcAuAuAAAcuAcAAGudTsdT A-96144.1 ACUUGuAGUUuAuAUGuAGdTsdT AD-45895.1 A-96177.1 GGGAGGcuuGAuGGAGAAudTsdT A-96178.1 AUUCUCcAUcAAGCCUCCCdTsdT AD-45902.1 A-96195.1 GGuGuuuucuAcuuGGGAudTsdT A-96196.1 AUCCcAAGuAGAAAAcACCdTsdT AD-45910.1 A-96229.1 AAGAGcAccAAGAAcuAcudTsdT A-96230.1 AGuAGUUCUUGGUGCUCUUdTsdT AD-45935.1 A-96239.1 uGGAGAAAAcAAccuAAAudTsdT A-96240.1 AUUuAGGUUGUUUUCUCcAdTsdT AD-45864.1 A-96151.1 GcAAcuAAcuAAcuuAAuudTsdT A-96152.1 AAUuAAGUuAGUuAGUUGCdTsdT AD-45873.1 A-96201.1 cAAccuAAAuGGuAAAuAudTsdT A-96202.1 AuAUUuACcAUUuAGGUUGdTsdT AD-45880.1 A-96125.1 GcuAuGuuAGAcGAuGuAAdTsdT A-96126.1 UuAcAUCGUCuAAcAuAGCdTsdT AD-45888.1 A-96159.1 cccAcAGAAAuuucucuAudTsdT A-96160.1 AuAGAGAAAUUUCUGUGGGdTsdT AD-45896.1 A-96193.1 GAuuuGGuGuuuucuAcuudTsdT A-96194.1 AAGuAGAAAAcACcAAAUCdTsdT AD-45903.1 A-96211.1 cAGAGccAAAAucAAGAuudTsdT A-96212.1 AAUCUUGAUUUUGGCUCUGdTsdT AD-45919.1 A-96217.1 AAAucAAGAuuuGcuAuGudTsdT A-96218.1 AcAuAGcAAAUCUUGAUUUdTsdT AD-45865.1 A-96167.1 cAuGGAcAuuAAuucAAcAdTsdT A-96168.1 UGUUGAAUuAAUGUCcAUGdTsdT AD-45874.1 A-96123.1 GAuuuGcuAuGuuAGAcGAdTsdT A-96124.1 UCGUCuAAcAuAGcAAAUCdTsdT AD-45881.1 A-96141.1 GAAcuAcAuAuAAAcuAcAdTsdT A-96142.1 UGuAGUUuAuAUGuAGUUCdTsdT AD-45889.1 A-96175.1 cGAAuAGAuGGAucAcAAAdTsdT A-96176.1 UUUGUGAUCcAUCuAUUCGdTsdT AD-45897.1 A-96209.1 cuuGuuAAAAcucuAAAcudTsdT A-96210.1 AGUUuAGAGUUUuAAcAAGdTsdT AD-45904.1 A-96227.1 AuuuGAucAGucuuuuuAudTsdT A-96228.1 AuAAAAAGACUGAUcAAAUdTsdT AD-45920.1 A-96233.1 uccAuGGAcAuuAAuucAAdTsdT A-96234.1 UUGAAUuAAUGUCcAUGGAdTsdT AD-45856.1 A-96117.1 cAcAAuuAAGcuccuucuudTsdT A-96118.1 AAGAAGGAGCUuAAUUGUGdTsdT AD-45929.1 A-96221.1 cAAcAuAuuuGAucAGucudTsdT A-96222.1 AGACUGAUcAAAuAUGUUGdTsdT AD-45866.1 A-96183.1 cuccAuAGuGAAGcAAucudTsdT A-96184.1 AGAUUGCUUcACuAUGGAGdTsdT AD-45875.1 A-96139.1 GccAAAuuAAuGAcAuAuudTsdT A-96140.1 AAuAUGUcAUuAAUUUGGCdTsdT AD-45882.1 A-96157.1 cAAcAGcAuAGucAAAuAAdTsdT A-96158.1 UuAUUUGACuAUGCUGUUGdTsdT AD-45890.1 A-96191.1 GGAAAucAcGAAAccAAcudTsdT A-96192.1 AGUUGGUUUCGUGAUUUCCdTsdT AD-45898.1 A-96131.1 cAGuuGGGAcAuGGucuuAdTsdT A-96132.1 uAAGACcAUGUCCcAACUGdTsdT AD-45857.1 A-96133.1 GAcAuGGucuuAAAGAcuudTsdT A-96134.1 AAGUCUUuAAGACcAUGUCdTsdT AD-45930.1 A-96237.1 uGuGGAGAAAAcAAccuAAdTsdT A-96238.1 UuAGGUUGUUUUCUCcAcAdTsdT AD-45867.1 A-96199.1 GuGGAGAAAAcAAccuAAAdTsdT A-96200.1 UUuAGGUUGUUUUCUCcACdTsdT AD-45876.1 A-96155.1 ccAAcAGcAuAGucAAAuAdTsdT A-96156.1 uAUUUGACuAUGCUGUUGGdTsdT AD-45883.1 A-96173.1 cAAcAucGAAuAGAuGGAudTsdT A-96174.1 AUCcAUCuAUUCGAUGUUGdTsdT AD-45891.1 A-96207.1 GcAAAuuuAAAAGGcAAuAdTsdT A-96208.1 uAUUGCCUUUuAAAUUUGCdTsdT AD-45914.1 A-96215.1 cAAAAucAAGAuuuGcuAudTsdT A-96216.1 AuAGcAAAUCUUGAUUUUGdTsdT AD-15838.1 A-26242.1 AGAGccAAAAucAAGAuuudTsdT A-26243.2 AAAUCUuGAUUUuGGCUCUdTsdT Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; s is a phosphothiorate linkage.

TABLE 4 Results of single dose screen using ANGPTL3 dsRNA sequences The experiments were conducted using modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD-15838.1. Delivery of siRNA duplexes was done using LNPs. Human Hep3B Duplex 10 nM 0.1 nM STDEV, 10 nM STDEV, 0.1 nM AD-15838.2 0.09 0.66 0.008 0.030 AD-45856.1 0.32 0.91 0.026 0.032 AD-45857.1 2.46 1.07 0.140 0.044 AD-45858.1 0.10 0.74 0.010 0.070 AD-45860.1 0.02 0.47 0.002 0.097 AD-45861.1 0.03 0.68 0.004 0.062 AD-45863.1 1.42 0.95 0.145 0.126 AD-45864.1 0.02 0.17 0.002 0.045 AD-45865.1 0.32 0.93 0.022 0.062 AD-45866.1 0.10 0.92 0.010 0.041 AD-45867.1 0.04 0.61 0.000 0.048 AD-45869.1 0.45 1.08 0.028 0.081 AD-45870.1 0.01 0.10 0.003 0.010 AD-45871.1 0.05 0.57 0.006 0.071 AD-45872.1 0.07 0.71 0.007 0.034 AD-45873.1 0.02 0.23 0.001 0.011 AD-45874.1 0.08 0.75 0.013 0.049 AD-45875.1 0.13 0.82 0.017 0.040 AD-45876.1 0.03 0.54 0.000 0.013 AD-45877.1 0.06 0.47 0.002 0.025 AD-45878.1 0.02 0.44 0.002 0.031 AD-45879.1 0.03 0.35 0.003 0.023 AD-45880.1 0.49 1.00 0.039 0.088 AD-45881.1 0.20 0.90 0.019 0.095 AD-45882.1 0.20 0.95 0.012 0.086 AD-45883.1 0.16 0.98 0.011 0.058 AD-45884.1 0.09 0.94 0.003 0.044 AD-45885.1 0.22 0.91 0.020 0.145 AD-45886.1 0.04 0.40 0.008 0.080 AD-45887.1 0.03 0.35 0.002 0.057 AD-45888.1 0.05 0.80 0.006 0.042 AD-45889.1 0.31 0.91 0.013 0.052 AD-45890.1 0.06 0.90 0.001 0.047 AD-45891.1 0.06 0.82 0.007 0.034 AD-45892.1 1.01 1.09 0.033 0.211 AD-45893.1 0.04 0.58 0.002 0.046 AD-45894.1 0.04 0.59 0.003 0.024 AD-45895.1 0.84 1.00 0.047 0.047 AD-45896.1 0.84 0.98 0.032 0.095 AD-45897.1 0.36 0.61 0.032 0.053 AD-45898.1 0.98 1.09 0.021 0.117 AD-45899.1 0.04 0.59 0.005 0.095 AD-45900.1 0.06 0.80 0.005 0.091 AD-45901.1 0.33 0.94 0.025 0.096 AD-45902.1 0.24 1.03 0.010 0.079 AD-45903.1 0.74 1.02 0.003 0.092 AD-45904.1 0.39 0.87 0.010 0.010 AD-45909.1 0.04 0.73 0.008 0.013 AD-45910.1 1.08 1.01 0.037 0.089 AD-45914.1 0.52 0.99 0.018 0.071 AD-45915.1 0.06 0.48 0.004 0.046 AD-45919.1 0.67 0.98 0.048 0.064 AD-45920.1 0.61 1.00 0.031 0.038 AD-45924.1 0.09 0.67 0.005 0.012 AD-45925.1 0.13 0.90 0.008 0.100 AD-45929.1 0.02 0.42 0.001 0.083 AD-45930.1 0.05 0.63 0.005 0.052 AD-45934.1 0.04 0.41 0.001 0.062 AD-45935.1 0.08 0.76 0.006 0.058 AD-45939.1 0.23 0.82 0.030 0.028 AD-1955.1 0.93 0.93 0.068 0.073 AD-1955.1 0.94 1.01 0.028 0.113 AD-1955.1 1.00 1.02 0.032 0.065 AD-1955.1 1.15 1.06 0.053 0.019

TABLE 5 Dose response screen results for ANGPTL3 dsRNA sequences The experiments were conducted using modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD-15838.1. Hep3B IC₅₀ 24 hrs 120 hrs IC₅₀ IC₅₀ IC₅₀ I IC₅₀ II weighted IC₅₀ I IC₅₀ II weighted Duplex (nM) (nM) (nM) (nM) (nM) (nM) AD-15838.2 0.027 0.006 0.017 0.657 0.937 0.800 AD-45860.1 0.006 0.002 0.004 0.045 0.032 0.039 AD-45864.1 0.002 0.001 0.002 0.046 0.042 0.044 AD-45870.1 0.002 0.001 0.001 0.011 0.008 0.010 AD-45873.1 0.005 0.004 0.005 0.037 0.025 0.031 AD-45876.1 0.032 0.006 0.019 0.269 0.045 0.156 AD-45877.1 0.018 0.012 0.015 1.660 0.538 1.091 AD-45878.1 0.023 0.015 0.019 0.252 0.131 0.190 AD-45879.1 0.002 0.003 0.003 0.023 0.029 0.026 AD-45886.1 0.004 0.004 0.004 0.030 0.018 0.025 AD-45887.1 0.010 0.009 0.010 0.058 0.059 0.059 AD-45915.1 0.016 0.015 0.015 0.110 0.056 0.083 AD-45929.1 0.023 0.008 0.016 0.227 0.025 0.124 AD-45934.1 0.006 0.006 0.006 0.110 0.045 0.077

TABLE 6 Results of cell viability screens using modified ANGPTL3 dsRNA sequences The experiments were conducted using modified oligonucleotide duplexes listed in Table 3. The sequence of AD-15838.2 is identical to the sequence of AD- 15838.1. Viability data is expressed as % viable relative to mock treated cells. Ave Ave Ave Ave Ave SD SD SD SD SD Target Duplex 10 nM 1 nM 500 pM 100 pM 50 pM 10 nM 1 nM 500 pM 100 pM 50 pM HeLa day 3 ANGPTL3 AD-15838.2 37.34 58.67 70.92 89.86 94.98 9.45 12.28 15.06 22.37 18.23 ANGPTL3 AD-15838.2 29.13 48.99 63.18 79.21 94.47 1.62 5.56 4.34 11.15 11.31 ANGPTL3 AD-45860.1 67.10 75.49 77.93 86.57 90.51 6.99 12.93 6.39 6.97 3.57 ANGPTL3 AD-45864.1 99.13 96.95 86.77 89.20 84.36 7.90 7.22 12.60 4.85 6.87 ANGPTL3 AD-45870.1 82.36 97.02 95.33 95.67 92.27 8.07 5.12 7.97 7.05 10.29 ANGPTL3 AD-45873.1 67.96 90.01 90.60 94.20 103.63 11.26 22.61 15.92 22.92 16.97 ANGPTL3 AD-45876.1 64.00 76.71 80.21 81.71 91.23 6.60 13.94 10.15 10.81 13.89 ANGPTL3 AD-45877.1 79.55 77.33 79.98 91.96 93.46 1.66 9.80 8.73 16.63 11.41 ANGPTL3 AD-45878.1 81.95 78.22 78.74 87.93 85.03 15.37 22.72 22.59 30.84 40.04 ANGPTL3 AD-45878.1 66.83 70.71 82.14 82.80 83.14 17.48 6.49 6.86 19.92 21.15 ANGPTL3 AD-45879.1 37.56 45.55 59.28 76.35 78.38 3.50 7.96 19.73 34.33 33.99 ANGPTL3 AD-45886.1 72.75 57.90 64.51 81.92 82.89 14.73 12.64 11.78 25.60 23.14 ANGPTL3 AD-45887.1 38.01 53.91 59.31 76.44 85.73 0.58 10.81 6.27 11.12 10.92 ANGPTL3 AD-45915.1 48.06 52.17 67.90 95.45 100.77 8.13 15.15 29.11 32.49 38.79 ANGPTL3 AD-45929.1 29.27 44.58 52.87 76.45 88.03 4.17 9.67 14.49 31.74 28.82 ANGPTL3 AD-45934.1 68.20 64.11 76.92 79.57 92.11 15.79 11.25 19.99 26.08 26.30 (+) control AD-19200 41.09 85.94 95.13 101.29 96.60 9.99 25.31 24.56 32.26 26.35 (+) control AD-19200 23.99 72.76 86.51 108.10 111.13 5.35 34.52 29.24 35.99 31.88 (−) control AD-1955 89.65 99.87 94.59 104.04 105.10 4.57 5.94 4.19 5.78 7.46 (−) control AD-1955 104.74 99.78 105.79 109.19 108.08 10.94 7.74 11.12 7.91 10.30 (−) control mock 100.00 6.92 (−) control mock 100.00 9.85 (+) control PLK 10.66 26.65 46.16 92.42 98.78 1.70 8.65 13.47 22.99 23.48 (+) control PLK 10.74 11.41 17.33 61.02 86.59 3.39 2.61 1.49 27.42 37.31 HeLa day 6 ANGPTL3 AD-15838.2 47.94 80.97 90.44 94.37 96.10 29.05 25.12 13.62 8.88 4.72 ANGPTL3 AD-15838.2 40.32 83.80 89.88 95.94 98.27 22.47 16.51 10.03 3.83 4.19 ANGPTL3 AD-45860.1 57.38 84.84 88.90 96.74 94.03 24.55 17.35 9.67 3.17 6.58 ANGPTL3 AD-45864.1 98.65 100.87 101.13 96.86 98.24 4.35 1.91 2.22 3.41 1.80 ANGPTL3 AD-45870.1 92.69 98.71 98.49 100.07 99.28 3.94 2.67 2.36 1.19 2.65 ANGPTL3 AD-45873.1 91.78 97.38 98.81 97.57 96.22 12.47 6.26 4.08 6.22 8.64 ANGPTL3 AD-45876.1 63.54 85.68 92.13 96.48 95.97 14.74 16.50 10.03 5.81 7.51 ANGPTL3 AD-45877.1 94.17 93.21 96.39 96.70 96.98 7.12 8.00 4.58 3.05 6.15 ANGPTL3 AD-45878.1 66.46 85.75 89.73 94.60 96.59 8.20 7.41 5.27 3.21 3.91 ANGPTL3 AD-45878.1 70.80 89.30 92.54 96.60 95.09 5.18 2.13 1.61 0.50 4.15 ANGPTL3 AD-45879.1 8.29 48.25 73.54 87.47 92.19 4.66 20.05 16.04 9.06 7.90 ANGPTL3 AD-45886.1 23.69 60.65 78.49 93.41 94.15 8.19 13.90 7.15 3.35 4.06 ANGPTL3 AD-45887.1 7.24 26.03 57.68 95.99 98.80 3.07 13.10 14.94 1.40 2.54 ANGPTL3 AD-45915.1 10.38 58.38 85.69 97.24 99.76 6.83 15.66 8.39 1.33 4.15 ANGPTL3 AD-45929.1 11.73 36.67 51.90 76.71 85.08 4.80 14.19 15.34 12.37 10.60 ANGPTL3 AD-45934.1 73.57 88.48 92.94 91.50 95.97 5.36 2.96 5.50 5.44 4.39 (+) control AD-19200 63.58 90.14 95.44 94.65 93.28 34.11 14.32 8.78 10.90 12.13 (+) control AD-19200 16.05 78.65 85.78 93.09 96.22 9.77 15.57 19.50 13.34 10.96 (−) control AD-1955 93.52 97.36 97.90 99.65 100.07 5.02 1.78 0.84 0.58 1.14 (−) control AD-1955 75.39 93.61 97.79 99.60 100.96 8.37 2.50 2.27 2.68 3.16 (−) control mock 100.00 1.32 (−) control mock 100.00 3.35 (+) control PLK 3.68 55.22 63.00 89.39 95.33 1.42 30.96 33.97 15.85 8.54 (+) control PLK 2.69 3.74 9.74 67.07 82.96 0.15 0.96 3.60 22.70 19.34 Hep3B day 3 ANGPTL3 AD-15838.2 35.33 61.00 68.79 82.74 90.41 2.41 6.21 4.21 2.61 7.07 ANGPTL3 AD-15838.2 35.34 61.04 72.14 89.71 106.88 1.49 2.61 7.37 6.48 7.13 ANGPTL3 AD-45860.1 17.79 39.25 60.57 94.28 99.85 1.07 3.51 3.57 13.09 16.41 ANGPTL3 AD-45864.1 80.35 88.19 87.01 89.39 92.09 6.93 6.98 9.42 7.41 17.05 ANGPTL3 AD-45870.1 75.00 93.30 96.64 106.29 99.08 7.10 12.24 4.01 5.95 9.64 ANGPTL3 AD-45873.1 42.68 78.45 82.26 97.11 96.58 5.17 5.04 8.31 12.11 11.33 ANGPTL3 AD-45876.1 31.37 55.00 70.69 93.49 91.00 4.39 6.09 5.47 15.11 6.38 ANGPTL3 AD-45877.1 74.45 94.60 96.70 103.77 106.75 3.27 2.44 3.45 6.10 7.40 ANGPTL3 AD-45878.1 50.22 69.65 80.49 92.77 97.37 2.51 14.94 10.44 8.21 5.30 ANGPTL3 AD-45878.1 44.85 65.39 75.67 92.83 109.67 10.10 7.76 8.56 7.78 4.97 ANGPTL3 AD-45879.1 23.73 60.81 84.59 95.72 108.68 6.43 21.36 19.62 13.69 5.95 ANGPTL3 AD-45886.1 27.19 55.35 64.97 100.18 102.09 0.97 6.65 11.46 6.91 4.08 ANGPTL3 AD-45887.1 41.70 97.18 101.91 111.27 105.18 9.26 6.81 7.36 1.72 2.23 ANGPTL3 AD-45915.1 45.10 66.31 82.22 97.97 103.30 6.91 11.84 14.79 6.54 2.48 ANGPTL3 AD-45929.1 48.58 79.14 89.96 95.00 101.37 10.40 10.29 10.52 18.24 10.53 ANGPTL3 AD-45934.1 80.15 102.93 112.82 114.16 113.98 5.28 0.62 4.19 0.75 3.99 (+) control AD-19200 14.79 55.23 72.90 89.64 94.30 2.17 5.42 7.19 10.28 16.39 (+) control AD-19200 22.76 92.02 101.56 106.68 113.09 6.61 18.99 7.41 9.83 10.64 (−) control AD-1955 77.77 81.25 82.23 88.21 95.02 2.83 5.40 5.08 5.42 6.63 (−) control AD-1955 80.42 86.70 90.23 93.46 97.04 10.53 5.70 8.14 3.27 3.45 (−) control mock 100.00 5.77 (−) control mock 100.00 9.79 (+) control PLK 10.91 12.89 14.31 23.87 50.93 0.17 0.87 1.64 1.13 7.80 (+) control PLK 13.19 16.12 22.89 55.03 94.35 0.78 0.88 8.36 18.88 9.85 Hep3B day 6 ANGPTL3 AD-15838.2 78.88 89.58 93.08 91.10 100.66 11.60 9.15 12.04 10.51 5.87 ANGPTL3 AD-15838.2 81.17 85.91 87.27 103.95 103.59 7.75 3.29 8.07 7.93 9.82 ANGPTL3 AD-45860.1 84.11 87.77 93.22 99.15 96.75 14.22 13.36 20.98 13.15 17.62 ANGPTL3 AD-45864.1 99.27 111.82 106.28 99.15 97.55 7.77 16.31 14.24 15.40 9.18 ANGPTL3 AD-45870.1 95.49 109.60 104.16 104.65 106.76 11.92 12.98 9.25 10.29 19.12 ANGPTL3 AD-45873.1 71.45 90.62 93.44 102.07 107.72 4.71 4.40 15.02 11.96 10.16 ANGPTL3 AD-45876.1 76.92 82.09 89.44 95.27 105.41 9.39 13.55 7.93 9.77 10.42 ANGPTL3 AD-45877.1 82.98 98.05 95.07 103.55 104.14 11.22 13.45 1.27 8.88 6.49 ANGPTL3 AD-45878.1 75.14 82.48 89.68 92.71 95.72 8.65 10.07 10.77 12.44 15.04 ANGPTL3 AD-45878.1 65.90 77.37 78.33 84.54 99.49 10.21 13.22 9.95 11.65 11.17 ANGPTL3 AD-45879.1 86.42 89.45 101.50 97.30 100.66 10.59 10.12 19.77 13.19 9.54 ANGPTL3 AD-45886.1 91.15 79.31 80.76 86.52 94.04 12.89 11.88 5.38 4.92 6.80 ANGPTL3 AD-45887.1 91.67 103.38 107.88 100.05 102.05 10.80 14.84 19.18 13.72 18.00 ANGPTL3 AD-45915.1 81.97 85.91 91.81 94.95 102.13 18.49 19.30 7.19 12.72 16.64 ANGPTL3 AD-45929.1 61.92 79.39 87.28 88.09 96.00 6.80 10.76 5.80 10.68 16.66 ANGPTL3 AD-45934.1 85.84 89.66 97.67 99.91 102.54 12.39 14.25 4.74 9.51 4.28 (+) control AD-19200 50.48 65.62 79.67 98.61 96.87 4.60 4.64 7.20 5.08 7.37 (+) control AD-19200 52.01 75.89 92.59 101.47 99.66 4.35 20.87 13.57 6.50 11.76 (−) control AD-1955 91.77 95.87 93.06 95.10 97.52 8.87 3.46 1.46 2.00 3.84 (−) control AD-1955 93.65 94.41 89.42 100.59 103.91 9.91 14.90 6.80 11.99 10.31 (−) control mock 100.00 5.10 (−) control mock 100.00 7.35 (+) control PLK 36.43 37.75 40.19 55.25 64.59 3.44 2.75 3.65 5.33 5.02 (+) control PLK 38.70 43.68 50.32 75.17 89.62 3.40 3.85 8.10 10.54 10.69

TABLE 7 Unmodified sense and antisense strand sequences of ANGPTL3 GalNac-conjugated dsRNAs Sense Antisense Sequence Sequence (SEQ ID NOS (SEQ ID NOS 264-448, 449-633, respectively, respectively, Sense in order of Position in Antisense in order of Position in Duplex ID Name appearance) NM_014495.2 Name appearance) NM_014495.2 AD-53063.1 A-108558.1 AAAGACAACAA 1066-1086 A-108559.1 AAUAUAAUGUUU 1064-1086 ACAUUAUAUUx GUUGUCUUUCC AD-52965.1 A-108310.1 ACAAUUAAGCU 58-78 A-108311.1 AAAAAGAAGGAG   56-78 CCUUCUUUUUx CUUAAUUGUGA AD-53030.1 A-108410.1 UGUCACUUGAA 398-418 A-108411.1 UUGAGUUGAGUU  396-418 CUCAACUCAAx CAAGUGACAUA AD-52953.1 A-108306.1 UCACAAUUAAG 56-76 A-108307.1 AAAGAAGGAGCU   54-76 CUCCUUCUUUx UAAUUGUGAAC AD-53001.1 A-108416.1 CUUGAACUCAA 403-423 A-108417.1 AAGUUUUGAGUU  401-423 CUCAAAACUUx GAGUUCAAGUG AD-53080.1 A-108548.1 CUCCAUAGUGA 1014-1034 A-108549.1 UUAGAUUGCUUC 1012-1034 AGCAAUCUAAx ACUAUGGAGUA AD-52971.1 A-108312.1 CAAUUAAGCUC 59-79 A-108313.1 UAAAAAGAAGGA   57-79 CUUCUUUUUAx GCUUAAUUGUG AD-53071.1 A-108498.1 ACCCAGCAACU 840-860 A-108499.1 AAAACUUGAGAG  838-860 CUCAAGUUUUx UUGCUGGGUCU AD-53024.1 A-108408.1 GAAUAUGUCAC 393-413 A-108409.1 UUGAGUUCAAGU  391-413 UUGAACUCAAx GACAUAUUCUU AD-52977.1 A-108314.1 AAUUAAGCUCC 60-80 A-108315.1 AUAAAAAGAAGG   58-80 UUCUUUUUAUx AGCUUAAUUGU AD-53064.1 A-108574.1 CAUUAUAUUGA 1078-1098 A-108575.1 AAAAGAAUAUUC 1076-1098 AUAUUCUUUUx AAUAUAAUGUU AD-53033.1 A-108458.1 ACUAACUAACU 483-503 A-108459.1 UUUUGAAUUAAG  481-503 UAAUUCAAAAx UUAGUUAGUUG AD-52954.1 A-108322.1 UUAUUGUUCCU 77-97 A-108323.1 AAAUAACUAGAG   75-97 CUAGUUAUUUx GAACAAUAAAA AD-53098.1 A-108554.1 CAUAGUGAAGC 1017-1037 A-108555.1 UAAUUAGAUUGC 1015-1037 AAUCUAAUUAx UUCACUAUGGA AD-53092.1 A-108552.1 CCAUAGUGAAG 1016-1036 A-108553.1 AAUUAGAUUGCU 1014-1036 CAAUCUAAUUx UCACUAUGGAG AD-53073.1 A-108530.1 GAUCACAAAAC 923-943 A-108531.1 UUUCAUUGAAGU  921-943 UUCAAUGAAAx UUUGUGAUCCA AD-53132.1 A-108628.1 AUGGAAGGUUA 1364-1384 A-108629.1 UUAUAGAGUAUA 1362-1384 UACUCUAUAAx ACCUUCCAUUU AD-53086.1 A-108550.1 UCCAUAGUGAA 1015-1035 A-108551.1 AUUAGAUUGCUU 1013-1035 GCAAUCUAAUx CACUAUGGAGU AD-52961.1 A-108340.1 CUAUGUUAGAC 164-184 A-108341.1 UUUUUACAUCGU  162-184 GAUGUAAAAAx CUAACAUAGCA AD-52983.1 A-108316.1 AUUAAGCUCCU 61-81 A-108317.1 AAUAAAAAGAAG   59-81 UCUUUUUAUUx GAGCUUAAUUG AD-53027.1 A-108456.1 AACUAACUAAC 482-502 A-108457.1 UUUGAAUUAAGU  480-502 UUAAUUCAAAx UAGUUAGUUGC AD-52986.1 A-108364.1 GGCCAAAUUAA 247-267 A-108365.1 AAAUAUGUCAUU  245-267 UGACAUAUUUx AAUUUGGCCCU AD-52989.1 A-108318.1 UUUUAUUGUUC 75-95 A-108319.1 AUAACUAGAGGA   73-95 CUCUAGUUAUx ACAAUAAAAAG AD-52981.1 A-108378.1 ACAUAUUUGAU 278-298 A-108379.1 AAAAAGACUGAU  276-298 CAGUCUUUUUx CAAAUAUGUUG AD-53077.1 A-108500.1 CCCAGCAACUC 841-861 A-108501.1 AAAAACUUGAGA  839-861 UCAAGUUUUUx GUUGCUGGGUC AD-53095.1 A-108506.1 CAGGUAGUCCA 884-904 A-108507.1 UUAAUGUCCAUG  882-904 UGGACAUUAAx GACUACCUGAU AD-52970.1 A-108390.1 ACUGAGAAGAA 345-365 A-108391.1 UUAUAUGUAGUU  343-365 CUACAUAUAAx CUUCUCAGUUC AD-53015.1 A-108452.1 GAGCAACUAAC 478-498 A-108453.1 AAUUAAGUUAGU  476-498 UAACUUAAUUx UAGUUGCUCUU AD-53147.1 A-108618.1 AACAACCUAAA 1282-1302 A-108619.1 AUAUUUACCAUU 1280-1302 UGGUAAAUAUx UAGGUUGUUUU AD-53103.1 A-108540.1 CCUAGAGAAGA  999-1019 A-108541.1 AUGGAGUAUAUC  997-1019 UAUACUCCAUx UUCUCUAGGCC AD-52969.1 A-108374.1 CAACAUAUUUG 276-296 A-108375.1 AAAGACUGAUCA  274-296 AUCAGUCUUUx AAUAUGUUGAG AD-53075.1 A-108562.1 ACAACAAACAU 1070-1090 A-108563.1 AUUCAAUAUAAU 1068-1090 UAUAUUGAAUx GUUUGUUGUCU AD-52994.1 A-108398.1 ACAUAUAAACU 358-378 A-108399.1 UUUGACUUGUAG  356-378 ACAAGUCAAAx UUUAUAUGUAG AD-52960.1 A-108324.1 CUAGUUAUUUC  88-108 A-108325.1 AAUUCUGGAGGA   86-108 CUCCAGAAUUx AAUAACUAGAG AD-53003.1 A-108448.1 AAGAGCAACUA 476-496 A-108449.1 UUAAGUUAGUUA  474-496 ACUAACUUAAx GUUGCUCUUCU AD-52995.1 A-108320.1 UUUAUUGUUCC 76-96 A-108321.1 AAUAACUAGAGG   74-96 UCUAGUUAUUx AACAAUAAAAA AD-53037.1 A-108428.1 CUCCUAGAAGA 430-450 A-108429.1 UAGAAUUUUUUC  428-450 AAAAAUUCUAx UUCUAGGAGGC AD-53087.1 A-108566.1 AACAAACAUUA 1072-1092 A-108567.1 AUAUUCAAUAUA 1070-1092 UAUUGAAUAUx AUGUUUGUUGU AD-53076.1 A-108578.1 GGAAAUCACGA 1105-1125 A-108579.1 AUAGUUGGUUUC 1103-1125 AACCAACUAUx GUGAUUUCCCA AD-52975.1 A-108376.1 AACAUAUUUGA 277-297 A-108377.1 AAAAGACUGAUC  275-297 UCAGUCUUUUx AAAUAUGUUGA AD-53138.1 A-108630.1 UGGAAGGUUAU 1365-1385 A-108631.1 UUUAUAGAGUAU 1363-1385 ACUCUAUAAAx AACCUUCCAUU AD-53091.1 A-108536.1 GGAGAACUACA 948-968 A-108537.1 AAACCAUAUUUG  946-968 AAUAUGGUUUx UAGUUCUCCCA AD-53124.1 A-108594.1 GAAAACAAAGA 1174-1194 A-108595.1 AAACACCAAAUC 1172-1194 UUUGGUGUUUx UUUGUUUUCCG AD-53125.1 A-108610.1 AGUGUGGAGAA 1271-1291 A-108611.1 UUAGGUUGUUUU 1269-1291 AACAACCUAAx CUCCACACUCA AD-53036.1 A-108412.1 GUCACUUGAAC 399-419 A-108413.1 UUUGAGUUGAGU  397-419 UCAACUCAAAx UCAAGUGACAU AD-53061.1 A-108526.1 GAUGGAUCACA 919-939 A-108527.1 AUUGAAGUUUUG  917-939 AAACUUCAAUx UGAUCCAUCUA AD-53093.1 A-108568.1 ACAAACAUUAU 1073-1093 A-108569.1 AAUAUUCAAUAU 1071-1093 AUUGAAUAUUx AAUGUUUGUUG AD-53137.1 A-108614.1 UGUGGAGAAAA 1273-1293 A-108615.1 AUUUAGGUUGUU 1271-1293 CAACCUAAAUx UUCUCCACACU AD-52999.1 A-108384.1 AUCAGUCUUUU 287-307 A-108385.1 AUAGAUCAUAAA  285-307 UAUGAUCUAUx AAGACUGAUCA AD-53069.1 A-108560.1 GACAACAAACA 1069-1089 A-108561.1 UUCAAUAUAAUG 1067-1089 UUAUAUUGAAx UUUGUUGUCUU AD-53034.1 A-108474.1 CAACAGCAUAG 622-642 A-108475.1 UUUUAUUUGACU  620-642 UCAAAUAAAAx AUGCUGUUGGU AD-52976.1 A-108392.1 CUGAGAAGAAC 346-366 A-108393.1 UUUAUAUGUAGU  344-366 UACAUAUAAAx UCUUCUCAGUU AD-52996.1 A-108336.1 UGCUAUGUUAG 162-182 A-108337.1 UUUACAUCGUCU  160-182 ACGAUGUAAAx AACAUAGCAAA AD-53029.1 A-108488.1 AACCCACAGAA 680-700 A-108489.1 AUAGAGAAAUUU  678-700 AUUUCUCUAUx CUGUGGGUUCU AD-53020.1 A-108438.1 CUUCAACAAAA 451-471 A-108439.1 AUAUUUCACUUU  449-471 AGUGAAAUAUx UUGUUGAAGUA AD-53042.1 A-108414.1 UCACUUGAACU 400-420 A-108415.1 UUUUGAGUUGAG  398-420 CAACUCAAAAx UUCAAGUGACA AD-53011.1 A-108482.1 CAUAGUCAAAU 628-648 A-108483.1 UAUUUCUUUUAU  626-648 AAAAGAAAUAx UUGACUAUGCU AD-52957.1 A-108370.1 CAAAAACUCAA 268-288 A-108371.1 AUCAAAUAUGUU  266-288 CAUAUUUGAUx GAGUUUUUGAA AD-53008.1 A-108434.1 UACUUCAACAA 449-469 A-108435.1 AUUUCACUUUUU  447-469 AAAGUGAAAUx GUUGAAGUAGA AD-53065.1 A-108496.1 GACCCAGCAAC 839-859 A-108497.1 AAACUUGAGAGU  837-859 UCUCAAGUUUx UGCUGGGUCUG AD-53115.1 A-108638.1 UUGAAUGAACU 1427-1447 A-108639.1 AAUUUGCCUCAG 1425-1447 GAGGCAAAUUx UUCAUUCAAAG AD-53012.1 A-108404.1 UAUAAACUACA 361-381 A-108405.1 AUUUUUGACUUG  359-381 AGUCAAAAAUx UAGUUUAUAUG AD-53004.1 A-108464.1 AAACAAGAUAA 559-579 A-108465.1 UUUGAUGCUAUU  557-579 UAGCAUCAAAx AUCUUGUUUUU AD-53021.1 A-108454.1 CAACUAACUAA 481-501 A-108455.1 UUGAAUUAAGUU  479-501 CUUAAUUCAAx AGUUAGUUGCU AD-52955.1 A-108338.1 GCUAUGUUAGA 163-183 A-108339.1 UUUUACAUCGUC  161-183 CGAUGUAAAAx UAACAUAGCAA AD-53119.1 A-108608.1 ACUUGGGAUCA 1198-1218 A-108609.1 UUUUGCUUUGUG 1196-1218 CAAAGCAAAAx AUCCCAAGUAG AD-52990.1 A-108334.1 UUGCUAUGUUA 161-181 A-108335.1 UUACAUCGUCUA  159-181 GACGAUGUAAx ACAUAGCAAAU AD-52964.1 A-108388.1 AACUGAGAAGA 344-364 A-108389.1 UAUAUGUAGUUC  342-364 ACUACAUAUAx UUCUCAGUUCC AD-52973.1 A-108344.1 GAUGUAAAAAU 175-195 A-108345.1 AUUGGCUAAAAU  173-195 UUUAGCCAAUx UUUUACAUCGU AD-53074.1 A-108546.1 ACUCCAUAGUG 1013-1033 A-108547.1 UAGAUUGCUUCA 1011-1033 AAGCAAUCUAx CUAUGGAGUAU AD-53026.1 A-108440.1 UUCAACAAAAA 452-472 A-108441.1 AAUAUUUCACUU  450-472 GUGAAAUAUUx UUUGUUGAAGU AD-53062.1 A-108542.1 CUAGAGAAGAU 1000-1020 A-108543.1 UAUGGAGUAUAU  998-1020 AUACUCCAUAx CUUCUCUAGGC AD-53114.1 A-108622.1 CAACCUAAAUG 1284-1304 A-108623.1 UUAUAUUUACCA 1282-1304 GUAAAUAUAAx UUUAGGUUGUU AD-53082.1 A-108580.1 GAAAUCACGAA 1106-1126 A-108581.1 UAUAGUUGGUUU 1104-1126 ACCAACUAUAx CGUGAUUUCCC AD-53035.1 A-108490.1 CCACAGAAAUU 683-703 A-108491.1 AAGAUAGAGAAA  681-703 UCUCUAUCUUx UUUCUGUGGGU AD-52978.1 A-108330.1 AAAUCAAGAUU 151-171 A-108331.1 UAACAUAGCAAA  149-171 UGCUAUGUUAx UCUUGAUUUUG AD-53084.1 A-108518.1 ACAUUAAUUCA 898-918 A-108519.1 UAUUCGAUGUUG  896-918 ACAUCGAAUAx AAUUAAUGUCC AD-52972.1 A-108328.1 CCAGAGCCAAA 142-162 A-108329.1 AAAUCUUGAUUU  140-162 AUCAAGAUUUx UGGCUCUGGAG AD-53002.1 A-108432.1 CUACUUCAACA 448-468 A-108433.1 UUUCACUUUUUG  446-468 AAAAGUGAAAx UUGAAGUAGAA AD-53078.1 A-108516.1 GACAUUAAUUC 897-917 A-108517.1 AUUCGAUGUUGA  895-917 AACAUCGAAUx AUUAAUGUCCA AD-53072.1 A-108514.1 GGACAUUAAUU 896-916 A-108515.1 UUCGAUGUUGAA  894-916 CAACAUCGAAx UUAAUGUCCAU AD-53005.1 A-108480.1 GCAUAGUCAAA 627-647 A-108481.1 AUUUCUUUUAUU  625-647 UAAAAGAAAUx UGACUAUGCUG AD-53083.1 A-108502.1 CUCUCAAGUUU 849-869 A-108503.1 UAGACAUGAAAA  847-869 UUCAUGUCUAx ACUUGAGAGUU AD-53102.1 A-108524.1 AUCGAAUAGAU 911-931 A-108525.1 UUUGUGAUCCAU  909-931 GGAUCACAAAx CUAUUCGAUGU AD-53105.1 A-108572.1 ACAUUAUAUUG 1077-1097 A-108573.1 AAAGAAUAUUCA 1075-1097 AAUAUUCUUUx AUAUAAUGUUU AD-53090.1 A-108520.1 UUAAUUCAACA 901-921 A-108521.1 AUCUAUUCGAUG  899-921 UCGAAUAGAUx UUGAAUUAAUG AD-53010.1 A-108466.1 GAUAAUAGCAU 565-585 A-108467.1 AAGGUCUUUGAU  563-585 CAAAGACCUUx GCUAUUAUCUU AD-52998.1 A-108368.1 UGACAUAUUUC 258-278 A-108369.1 UUGAGUUUUUGA  256-278 AAAAACUCAAx AAUAUGUCAUU AD-52992.1 A-108366.1 AAAUUAAUGAC 251-271 A-108367.1 UUUGAAAUAUGU  249-271 AUAUUUCAAAx CAUUAAUUUGG AD-53068.1 A-108544.1 GAAGAUAUACU 1005-1025 A-108545.1 UUCACUAUGGAG 1003-1025 CCAUAGUGAAx UAUAUCUUCUC AD-53032.1 A-108442.1 AAUAUUUAGAA 467-487 A-108443.1 UUAGUUGCUCUU  465-487 GAGCAACUAAx CUAAAUAUUUC AD-52967.1 A-108342.1 CGAUGUAAAAA 174-194 A-108343.1 UUGGCUAAAAUU  172-194 UUUUAGCCAAx UUUACAUCGUC AD-53096.1 A-108522.1 UUCAACAUCGA 905-925 A-108523.1 AUCCAUCUAUUC  903-925 AUAGAUGGAUx GAUGUUGAAUU AD-53131.1 A-108612.1 GUGUGGAGAAA 1272-1292 A-108613.1 UUUAGGUUGUUU 1270-1292 ACAACCUAAAx UCUCCACACUC AD-52963.1 A-108372.1 UCAACAUAUUU 275-295 A-108373.1 AAGACUGAUCAA  273-295 GAUCAGUCUUx AUAUGUUGAGU AD-53089.1 A-108504.1 UCAGGUAGUCC 883-903 A-108505.1 UAAUGUCCAUGG  881-903 AUGGACAUUAx ACUACCUGAUA AD-53044.1 A-108446.1 UUUAGAAGAGC 471-491 A-108447.1 UUAGUUAGUUGC  469-491 AACUAACUAAx UCUUCUAAAUA AD-52988.1 A-108396.1 UACAUAUAAAC 357-377 A-108397.1 UUGACUUGUAGU  355-377 UACAAGUCAAx UUAUAUGUAGU AD-53067.1 A-108528.1 GGAUCACAAAA 922-942 A-108529.1 UUCAUUGAAGUU  920-942 CUUCAAUGAAx UUGUGAUCCAU AD-53009.1 A-108450.1 AGAGCAACUAA 477-497 A-108451.1 AUUAAGUUAGUU  475-497 CUAACUUAAUx AGUUGCUCUUC AD-53022.1 A-108470.1 ACCAACAGCAU 620-640 A-108471.1 UUAUUUGACUAU  618-640 AGUCAAAUAAx GCUGUUGGUUU AD-53016.1 A-108468.1 AACCAACAGCA 619-639 A-108469.1 UAUUUGACUAUG  617-639 UAGUCAAAUAx CUGUUGGUUUA AD-53007.1 A-108418.1 GAACUCAACUC 406-426 A-108419.1 UUCAAGUUUUGA  404-426 AAAACUUGAAx GUUGAGUUCAA AD-53148.1 A-108634.1 UACUCUAUAAA 1375-1395 A-108635.1 UUUGGUUGAUUU 1373-1395 AUCAACCAAAx UAUAGAGUAUA AD-53040.1 A-108476.1 CAGCAUAGUCA 625-645 A-108477.1 UUCUUUUAUUUG  623-645 AAUAAAAGAAx ACUAUGCUGUU AD-53041.1 A-108492.1 GAAAUAAGAAA 748-768 A-108493.1 AUGUUUUACAUU  746-768 UGUAAAACAUx UCUUAUUUCAU AD-53039.1 A-108460.1 CUAACUAACUU 484-504 A-108461.1 AUUUUGAAUUAA  482-504 AAUUCAAAAUx GUUAGUUAGUU AD-53139.1 A-108646.1 AUGAACUGAGG 1431-1451 A-108647.1 UUUAAAUUUGCC 1429-1451 CAAAUUUAAAx UCAGUUCAUUC AD-53144.1 A-108648.1 UGAACUGAGGC 1432-1452 A-108649.1 UUUUAAAUUUGC 1430-1452 AAAUUUAAAAx CUCAGUUCAUU AD-53142.1 A-108616.1 AAACAACCUAA 1281-1301 A-108617.1 UAUUUACCAUUU 1279-1301 AUGGUAAAUAx AGGUUGUUUUC AD-53108.1 A-108620.1 ACAACCUAAAU 1283-1303 A-108621.1 UAUAUUUACCAU 1281-1303 GGUAAAUAUAx UUAGGUUGUUU AD-53079.1 A-108532.1 AACGUGGGAGA 942-962 A-108533.1 UAUUUGUAGUUC  940-962 ACUACAAAUAx UCCCACGUUUC AD-53133.1 A-108644.1 AAUGAACUGAG 1430-1450 A-108645.1 UUAAAUUUGCCU 1428-1450 GCAAAUUUAAx CAGUUCAUUCA AD-53104.1 A-108556.1 GUUGGAAGACU 1053-1073 A-108557.1 UUGUCUUUCCAG 1051-1073 GGAAAGACAAx UCUUCCAACUC AD-53088.1 A-108582.1 UGGCAAUGUCC 1149-1169 A-108583.1 AUUGCAUUGGGG 1147-1169 CCAAUGCAAUx ACAUUGCCAGU AD-53101.1 A-108508.1 GGUAGUCCAUG 886-906 A-108509.1 AAUUAAUGUCCA  884-906 GACAUUAAUUx UGGACUACCUG AD-53000.1 A-108400.1 CAUAUAAACUA 359-379 A-108401.1 UUUUGACUUGUA  357-379 CAAGUCAAAAx GUUUAUAUGUA AD-53112.1 A-108590.1 AAUCCCGGAAA 1167-1187 A-108591.1 AAAUCUUUGUUU 1165-1187 ACAAAGAUUUx UCCGGGAUUGC AD-53107.1 A-108604.1 CUACUUGGGAU 1196-1216 A-108605.1 UUGCUUUGUGAU 1194-1216 CACAAAGCAAx CCCAAGUAGAA AD-53121.1 A-108640.1 UGAAUGAACUG 1428-1448 A-108641.1 AAAUUUGCCUCA 1426-1448 AGGCAAAUUUx GUUCAUUCAAA AD-53046.1 A-108478.1 AGCAUAGUCAA 626-646 A-108479.1 UUUCUUUUAUUU  624-646 AUAAAAGAAAx GACUAUGCUGU AD-53038.1 A-108444.1 AUUUAGAAGAG 470-490 A-108445.1 UAGUUAGUUGCU  468-490 CAACUAACUAx CUUCUAAAUAU AD-53140.1 A-108662.1 AGGCAAAUUUA 1439-1459 A-108663.1 UAUUGCCUUUUA 1437-1459 AAAGGCAAUAx AAUUUGCCUCA AD-52987.1 A-108380.1 CAUAUUUGAUC 279-299 A-108381.1 UAAAAAGACUGA  277-299 AGUCUUUUUAx UCAAAUAUGUU AD-53130.1 A-108596.1 AAAACAAAGAU 1175-1195 A-108597.1 AAAACACCAAAU 1173-1195 UUGGUGUUUUx CUUUGUUUUCC AD-53106.1 A-108588.1 CAAUCCCGGAA 1166-1186 A-108589.1 AAUCUUUGUUUU 1164-1186 AACAAAGAUUx CCGGGAUUGCA AD-53081.1 A-108564.1 CAACAAACAUU 1071-1091 A-108565.1 UAUUCAAUAUAA 1069-1091 AUAUUGAAUAx UGUUUGUUGUC AD-53118.1 A-108592.1 GGAAAACAAAG 1173-1193 A-108593.1 AACACCAAAUCU 1171-1193 AUUUGGUGUUx UUGUUUUCCGG AD-53136.1 A-108598.1 ACAAAGAUUUG 1178-1198 A-108599.1 UAGAAAACACCA 1176-1198 GUGUUUUCUAx AAUCUUUGUUU AD-53127.1 A-108642.1 GAAUGAACUGA 1429-1449 A-108643.1 UAAAUUUGCCUC 1427-1449 GGCAAAUUUAx AGUUCAUUCAA AD-53066.1 A-108512.1 CCAUGGACAUU 892-912 A-108513.1 AUGUUGAAUUAA  890-912 AAUUCAACAUx UGUCCAUGGAC AD-53013.1 A-108420.1 AACUCAACUCA 407-427 A-108421.1 UUUCAAGUUUUG  405-427 AAACUUGAAAx AGUUGAGUUCA AD-52991.1 A-108350.1 CAGUUGGGACA 205-225 A-108351.1 UUUAAGACCAUG  203-225 UGGUCUUAAAx UCCCAACUGAA AD-53099.1 A-108570.1 AACAUUAUAUU 1076-1096 A-108571.1 AAGAAUAUUCAA 1074-1096 GAAUAUUCUUx UAUAAUGUUUG AD-52958.1 A-108386.1 ACCAGUGAAAU 316-336 A-108387.1 UUCUUCUUUGAU  314-336 CAAAGAAGAAx UUCACUGGUUU AD-53097.1 A-108538.1 GUUGGGCCUAG  993-1013 A-108539.1 UAUAUCUUCUCU  991-1013 AGAAGAUAUAx AGGCCCAACCA AD-52966.1 A-108326.1 CUCCAGAGCCA 140-160 A-108327.1 AUCUUGAUUUUG  138-160 AAAUCAAGAUx GCUCUGGAGAU AD-53145.1 A-108664.1 GGCAAAUUUAA 1440-1460 A-108665.1 UUAUUGCCUUUU 1438-1460 AAGGCAAUAAx AAAUUUGCCUC AD-53113.1 A-108606.1 UACUUGGGAUC 1197-1217 A-108607.1 UUUGCUUUGUGA 1195-1217 ACAAAGCAAAx UCCCAAGUAGA AD-52993.1 A-108382.1 GAUCAGUCUUU 286-306 A-108383.1 UAGAUCAUAAAA  284-306 UUAUGAUCUAx AGACUGAUCAA AD-53031.1 A-108426.1 GAAAGCCUCCU 424-444 A-108427.1 UUUUUCUUCUAG  422-444 AGAAGAAAAAx GAGGCUUUCAA AD-53017.1 A-108484.1 AGUCAAAUAAA 631-651 A-108485.1 UUCUAUUUCUUU  629-651 AGAAAUAGAAx UAUUUGACUAU AD-53143.1 A-108632.1 AUACUCUAUAA 1374-1394 A-108633.1 UUGGUUGAUUUU 1372-1394 AAUCAACCAAx AUAGAGUAUAA AD-53149.1 A-108650.1 GAACUGAGGCA 1433-1453 A-108651.1 UUUUUAAAUUUG 1431-1453_G21A AAUUUAAAAAx CCUCAGUUCAU AD-53059.1 A-108494.1 AGACCCAGCAA 838-858 A-108495.1 AACUUGAGAGUU  836-858 CUCUCAAGUUx GCUGGGUCUGA AD-53006.1 A-108402.1 AUAUAAACUAC 360-380 A-108403.1 UUUUUGACUUGU  358-380 AAGUCAAAAAx AGUUUAUAUGU AD-53025.1 A-108424.1 UGAAAGCCUCC 423-443 A-108425.1 UUUUCUUCUAGG  421-443 UAGAAGAAAAx AGGCUUUCAAG AD-53085.1 A-108534.1 GGGAGAACUAC 947-967 A-108535.1 AACCAUAUUUGU  945-967 AAAUAUGGUUx AGUUCUCCCAC AD-52984.1 A-108332.1 AGAUUUGCUAU 157-177 A-108333.1 AUCGUCUAACAU  155-177 GUUAGACGAUx AGCAAAUCUUG AD-53023.1 A-108486.1 GAACCCACAGA 679-699 A-108487.1 UAGAGAAAUUUC  677-699 AAUUUCUCUAx UGUGGGUUCUU AD-53014.1 A-108436.1 ACUUCAACAAA 450-470 A-108437.1 UAUUUCACUUUU  448-470 AAGUGAAAUAx UGUUGAAGUAG AD-53060.1 A-108510.1 AGUCCAUGGAC 889-909 A-108511.1 UUGAAUUAAUGU  887-909 AUUAAUUCAAx CCAUGGACUAC AD-53110.1 A-108652.1 AACUGAGGCAA 1434-1454 A-108653.1 UCUUUUAAAUUU 1432-1454_G21A AUUUAAAAGAx GCCUCAGUUCA AD-52980.1 A-108362.1 GGGCCAAAUUA 246-266 A-108363.1 AAUAUGUCAUUA  244-266 AUGACAUAUUx AUUUGGCCCUU AD-53109.1 A-108636.1 AUCCAUCCAAC 1402-1422 A-108637.1 UUCUGAAUCUGU 1400-1422 AGAUUCAGAAx UGGAUGGAUCA AD-53141.1 A-108600.1 AAGAUUUGGUG 1181-1201 A-108601.1 AAGUAGAAAACA 1179-1201 UUUUCUACUUx CCAAAUCUUUG AD-53126.1 A-108626.1 GUCUCAAAAUG 1356-1376 A-108627.1 UAUAACCUUCCA 1354-1376 GAAGGUUAUAx UUUUGAGACUU AD-53116.1 A-108654.1 ACUGAGGCAAA 1435-1455 A-108655.1 UCCUUUUAAAUU 1433-1455_C21A UUUAAAAGGAx UGCCUCAGUUC AD-52997.1 A-108352.1 GGGACAUGGUC 210-230 A-108353.1 AAGUCUUUAAGA  208-230 UUAAAGACUUx CCAUGUCCCAA AD-53120.1 A-108624.1 AUGGUAAAUAU 1292-1312 A-108625.1 UUGGUUUGUUAU 1290-1312 AACAAACCAAx AUUUACCAUUU AD-53070.1 A-108576.1 GGGAAAUCACG 1104-1124 A-108577.1 UAGUUGGUUUCG 1102-1124 AAACCAACUAx UGAUUUCCCAA AD-53028.1 A-108472.1 CCAACAGCAUA 621-641 A-108473.1 UUUAUUUGACUA  619-641 GUCAAAUAAAx UGCUGUUGGUU AD-53146.1 A-108602.1 UUUUCUACUUG 1192-1212 A-108603.1 UUUGUGAUCCCA 1190-1212 GGAUCACAAAx AGUAGAAAACA AD-52982.1 A-108394.1 AGAACUACAUA 352-372 A-108395.1 UUGUAGUUUAUA  350-372 UAAACUACAAx UGUAGUUCUUC AD-53111.1 A-108668.1 AGAGUAUGUGU 1915-1935 A-108669.1 ACAGAUUUUUAC 1913-1935 AAAAAUCUGUx ACAUACUCUGU AD-53045.1 A-108462.1 AAAACAAGAUA 558-578 A-108463.1 UUGAUGCUAUUA  556-578 AUAGCAUCAAx UCUUGUUUUUC AD-53123.1 A-108672.1 AGUAUGUGUAA 1917-1937 A-108673.1 UUACAGAUUUUU 1915-1937 AAAUCUGUAAx ACACAUACUCU AD-53018.1 A-108406.1 AGUCAAAAAUG 372-392 A-108407.1 UUUACCUCUUCA  370-392 AAGAGGUAAAx UUUUUGACUUG AD-52956.1 A-108354.1 GGACAUGGUCU 211-231 A-108355.1 AAAGUCUUUAAG  209-231 UAAAGACUUUx ACCAUGUCCCA AD-53134.1 A-108660.1 GAGGCAAAUUU 1438-1458 A-108661.1 AUUGCCUUUUAA 1436-1458 AAAAGGCAAUx AUUUGCCUCAG AD-52968.1 A-108358.1 GUCUUAAAGAC 218-238 A-108359.1 UAUGGACAAAGU  216-238 UUUGUCCAUAx CUUUAAGACCA AD-53122.1 A-108656.1 CUGAGGCAAAU 1436-1456 A-108657.1 UGCCUUUUAAAU 1434-1456 UUAAAAGGCAx UUGCCUCAGUU AD-53100.1 A-108586.1 GCAAUCCCGGA 1165-1185 A-108587.1 AUCUUUGUUUUC 1163-1185 AAACAAAGAUx CGGGAUUGCAU AD-53128.1 A-108658.1 UGAGGCAAAUU 1437-1457 A-108659.1 UUGCCUUUUAAA 1435-1457 UAAAAGGCAAx UUUGCCUCAGU AD-53043.1 A-108430.1 UCUACUUCAAC 447-467 A-108431.1 UUCACUUUUUGU  445-467 AAAAAGUGAAx UGAAGUAGAAU AD-53135.1 A-108676.1 UAUGUGUAAAA 1919-1939 A-108677.1 UAUUACAGAUUU 1917-1939 AUCUGUAAUAx UUACACAUACU AD-53094.1 A-108584.1 AAUGCAAUCCC 1162-1182 A-108585.1 UUUGUUUUCCGG 1160-1182 GGAAAACAAAx GAUUGCAUUGG AD-53019.1 A-108422.1 CUUGAAAGCCU 421-441 A-108423.1 UUCUUCUAGGAG  419-441 CCUAGAAGAAx GCUUUCAAGUU AD-53129.1 A-108674.1 GUAUGUGUAAA 1918-1938 A-108675.1 AUUACAGAUUUU 1916-1938 AAUCUGUAAUx UACACAUACUC AD-53150.1 A-108666.1 CAGAGUAUGUG 1914-1934 A-108667.1 AAGAUUUUUACA 1912-1934_G21U UAAAAAUCUUx CAUACUCUGUG AD-53117.1 A-108670.1 GAGUAUGUGUA 1916-1936 A-108671.1 UACAGAUUUUUA 1914-1936 AAAAUCUGUAx CACAUACUCUG AD-52985.1 A-108348.1 UCAGUUGGGAC 204-224 A-108349.1 UUAAGACCAUGU  202-224 AUGGUCUUAAx CCCAACUGAAG AD-52962.1 A-108356.1 GGUCUUAAAGA 217-237 A-108357.1 AUGGACAAAGUC  215-237 CUUUGUCCAUx UUUAAGACCAU AD-52974.1 A-108360.1 UCUUAAAGACU 219-239 A-108361.1 UUAUGGACAAAG  217-239 UUGUCCAUAAx UCUUUAAGACC AD-52979.1 A-108346.1 UUCAGUUGGGA 203-223 A-108347.1 UAAGACCAUGUC  201-223 CAUGGUCUUAx CCAACUGAAGG The symbol “x” indicates that the sequence contains a GalNAc conjugate.

TABLE 8 Modified sense and antisense strand sequences of ANGPTL3 GalNac-conjugated dsRNAs Sense Sequence Antisense Sequence (SEQ ID NOS 634-818, (SEQ ID NOS 819-1003, Sense respectively, in Antisense respectively, in Duplex ID OligoName order of appearance) OligoName order of appearance) AD-53063.1 A-108558.1 AfaAfgAfcAfaCfAfAfaC A-108559.1 aAfuAfuAfaUfgUfuugUfu faUfuAfuAfuUfL96 GfuCfuUfusCfsc AD-52965.1 A-108310.1 AfcAfaUfuAfaGfCfUfcC A-108311.1 aAfaAfaGfaAfgGfagcUfu fuUfcUfuUfuUfL96 AfaUfuGfusGfsa AD-53030.1 A-108410.1 UfgUfcAfcUfuGfAfAfcU A-108411.1 uUfgAfgUfuGfaGfuuc fcAfaCfuCfaAfL96 AfaGfuGfaCfasUfsa AD-52953.1 A-108306.1 UfcAfcAfaUfuAfAfGfcU A-108307.1 aAfaGfaAfgGfaGfcuuAfa fcCfuUfcUfuUfL96 UfuGfuGfasAfsc AD-53001.1 A-108416.1 CfuUfgAfaCfuCfAfAfcU A-108417.1 aAfgUfuUfuGfaGfuugAfg fcAfaAfaCfuUfL96 UfuCfaAfgsUfsg AD-53080.1 A-108548.1 CfuCfcAfuAfgUfGfAfaG A-108549.1 uUfaGfaUfuGfcUfucaCfu fcAfaUfcUfaAfL96 AfuGfgAfgsUfsa AD-52971.1 A-108312.1 CfaAfuUfaAfgCfUfCfcU A-108313.1 uAfaAfaAfgAfaGfgagCfu fuCfuUfuUfuAfL96 UfaAfuUfgsUfsg AD-53071.1 A-108498.1 AfcCfcAfgCfaAfCfUfcU A-108499.1 aAfaAfcUfuGfaGfaguUfg fcAfaGfuUfuUfL96 CfuGfgGfusCfsu AD-53024.1 A-108408.1 GfaAfuAfuGfuCfAfCfuU A-108409.1 uUfgAfgUfuCfaAfgugAfc fgAfaCfuCfaAfL96 AfuAfuUfcsUfsu AD-52977.1 A-108314.1 AfaUfuAfaGfcUfCfCfuU A-108315.1 aUfaAfaAfaGfaAfggaGfc fcUfuUfuUfaUfL96 UfuAfaUfusGfsu AD-53064.1 A-108574.1 CfaUfuAfuAfuUfGfAfaU A-108575.1 aAfaAfgAfaUfaUfucaAfu faUfuCfuUfuUfL96 AfuAfaUfgsUfsu AD-53033.1 A-108458.1 AfcUfaAfcUfaAfCfUfuA A-108459.1 uUfuUfgAfaUfuAfaguUfa faUfuCfaAfaAfL96 GfuUfaGfusUfsg AD-52954.1 A-108322.1 UfuAfuUfgUfuCfCfUfcU A-108323.1 aAfaUfaAfcUfaGfaggAfa faGfuUfaUfuUfL96 CfaAfuAfasAfsa AD-53098.1 A-108554.1 CfaUfaGfuGfaAfGfCfaA A-108555.1 uAfaUfuAfgAfuUfgcuUfc fuCfuAfaUfuAfL96 AfcUfaUfgsGfsa AD-53092.1 A-108552.1 CfcAfuAfgUfgAfAfGfcA A-108553.1 aAfuUfaGfaUfuGfcuuCfa faUfcUfaAfuUfL96 CfuAfuGfgsAfsg AD-53073.1 A-108530.1 GfaUfcAfcAfaAfAfCfuU A-108531.1 uUfuCfaUfuGfaAfguuUfu fcAfaUfgAfaAfL96 GfuGfaUfcsCfsa AD-53132.1 A-108628.1 AfuGfgAfaGfgUfUfAfuA A-108629.1 uUfaUfaGfaGfuAfuaaCfc fcUfcUfaUfaAfL96 UfuCfcAfusUfsu AD-53086.1 A-108550.1 UfcCfaUfaGfuGfAfAfgC A-108551.1 aUfuAfgAfuUfgCfuucAfc faAfuCfuAfaUfL96 UfaUfgGfasGfsu AD-52961.1 A-108340.1 CfuAfuGfuUfaGfAfCfgA A-108341.1 uUfuUfuAfcAfuCfgucUfa fuGfuAfaAfaAfL96 AfcAfuAfgsCfsa AD-52983.1 A-108316.1 AfuUfaAfgCfuCfCfUfuC A-108317.1 aAfuAfaAfaAfgAfaggAfg fuUfuUfuAfuUfL96 CfuUfaAfusUfsg AD-53027.1 A-108456.1 AfaCfuAfaCfuAfAfCfuU A-108457.1 uUfuGfaAfuUfaAfguuAfg faAfuUfcAfaAfL96 UfuAfgUfusGfsc AD-52986.1 A-108364.1 GfgCfcAfaAfuUfAfAfuG A-108365.1 aAfaUfaUfgUfcAfuuaAfu faCfaUfaUfuUfL96 UfuGfgCfcsCfsu AD-52989.1 A-108318.1 UfuUfuAfuUfgUfUfCfcU A-108319.1 aUfaAfcUfaGfaGfgaaCfa fcUfaGfuUfaUfL96 AfuAfaAfasAfsg AD-52981.1 A-108378.1 AfcAfuAfuUfuGfAfUfcA A-108379.1 aAfaAfaGfaCfuGfaucAfa fgUfcUfuUfuUfL96 AfuAfuGfusUfsg AD-53077.1 A-108500.1 CfcCfaGfcAfaCfUfCfuC A-108501.1 aAfaAfaCfuUfgAfgagUfu faAfgUfuUfuUfL96 GfcUfgGfgsUfsc AD-53095.1 A-108506.1 CfaGfgUfaGfuCfCfAfuG A-108507.1 uUfaAfuGfuCfcAfuggAfc fgAfcAfuUfaAfL96 UfaCfcUfgsAfsu AD-52970.1 A-108390.1 AfcUfgAfgAfaGfAfAfcU A-108391.1 uUfaUfaUfgUfaGfuucUfu faCfaUfaUfaAfL96 CfuCfaGfusUfsc AD-53015.1 A-108452.1 GfaGfcAfaCfuAfAfCfuA A-108453.1 aAfuUfaAfgUfuAfguuAfg faCfuUfaAfuUfL96 UfuGfcUfcsUfsu AD-53147.1 A-108618.1 AfaCfaAfcCfuAfAfAfuG A-108619.1 aUfaUfuUfaCfcAfuuuAfg fgUfaAfaUfaUfL96 GfuUfgUfusUfsu AD-53103.1 A-108540.1 CfcUfaGfaGfaAfGfAfuA A-108541.1 aUfgGfaGfuAfuAfucuUfc fuAfcUfcCfaUfL96 UfcUfaGfgsCfsc AD-52969.1 A-108374.1 CfaAfcAfuAfuUfUfGfaU A-108375.1 aAfaGfaCfuGfaUfcaaAfu fcAfgUfcUfuUfL96 AfuGfuUfgsAfsg AD-53075.1 A-108562.1 AfcAfaCfaAfaCfAfUfuA A-108563.1 aUfuCfaAfuAfuAfaugUfu fuAfuUfgAfaUfL96 UfgUfuGfusCfsu AD-52994.1 A-108398.1 AfcAfuAfuAfaAfCfUfaC A-108399.1 uUfuGfaCfuUfgUfaguUfu faAfgUfcAfaAfL96 AfuAfuGfusAfsg AD-52960.1 A-108324.1 CfuAfgUfuAfuUfUfCfcU A-108325.1 aAfuUfcUfgGfaGfgaaAfu fcCfaGfaAfuUfL96 AfaCfuAfgsAfsg AD-53003.1 A-108448.1 AfaGfaGfcAfaCfUfAfaC A-108449.1 uUfaAfgUfuAfgUfuagUfu fuAfaCfuUfaAfL96 GfcUfcUfusCfsu AD-52995.1 A-108320.1 UfuUfaUfuGfuUfCfCfuC A-108321.1 aAfuAfaCfuAfgAfggaAfc fuAfgUfuAfuUfL96 AfaUfaAfasAfsa AD-53037.1 A-108428.1 CfuCfcUfaGfaAfGfAfaA A-108429.1 uAfgAfaUfuUfuUfucuUfc faAfaUfuCfuAfL96 UfaGfgAfgsGfsc AD-53087.1 A-108566.1 AfaCfaAfaCfaUfUfAfuA A-108567.1 aUfaUfuCfaAfuAfuaaUfg fuUfgAfaUfaUfL96 UfuUfgUfusGfsu AD-53076.1 A-108578.1 GfgAfaAfuCfaCfGfAfaA A-108579.1 aUfaGfuUfgGfuUfucgUfg fcCfaAfcUfaUfL96 AfuUfuCfcsCfsa AD-52975.1 A-108376.1 AfaCfaUfaUfuUfGfAfuC A-108377.1 aAfaAfgAfcUfgAfucaAfa faGfuCfuUfuUfL96 UfaUfgUfusGfsa AD-53138.1 A-108630.1 UfgGfaAfgGfuUfAfUfaC A-108631.1 uUfuAfuAfgAfgUfauaAfc fuCfuAfuAfaAfL96 CfuUfcCfasUfsu AD-53091.1 A-108536.1 GfgAfgAfaCfuAfCfAfaA A-108537.1 aAfaCfcAfuAfuUfuguAfg fuAfuGfgUfuUfL96 UfuCfuCfcsCfsa AD-53124.1 A-108594.1 GfaAfaAfcAfaAfGfAfuU A-108595.1 aAfaCfaCfcAfaAfucuUfu fuGfgUfgUfuUfL96 GfuUfuUfcsCfsg AD-53125.1 A-108610.1 AfgUfgUfgGfaGfAfAfaA A-108611.1 uUfaGfgUfuGfuUfuucUfc fcAfaCfcUfaAfL96 CfaCfaCfusCfsa AD-53036.1 A-108412.1 GfuCfaCfuUfgAfAfCfuC A-108413.1 uUfuGfaGfuUfgAfguuCfa faAfcUfcAfaAfL96 AfgUfgAfcsAfsu AD-53061.1 A-108526.1 GfaUfgGfaUfcAfCfAfaA A-108527.1 aUfuGfaAfgUfuUfuguGfa faCfuUfcAfaUfL96 UfcCfaUfcsUfsa AD-53093.1 A-108568.1 AfcAfaAfcAfuUfAfUfaU A-108569.1 aAfuAfuUfcAfaUfauaAfu fuGfaAfuAfuUfL96 GfuUfuGfusUfsg AD-53137.1 A-108614.1 UfgUfgGfaGfaAfAfAfcA A-108615.1 aUfuUfaGfgUfuGfuuuUfc faCfcUfaAfaUfL96 UfcCfaCfasCfsu AD-52999.1 A-108384.1 AfuCfaGfuCfuUfUfUfuA A-108385.1 aUfaGfaUfcAfuAfaaaAfg fuGfaUfcUfaUfL96 AfcUfgAfusCfsa AD-53069.1 A-108560.1 GfaCfaAfcAfaAfCfAfuU A-108561.1 uUfcAfaUfaUfaAfuguUfu faUfaUfuGfaAfL96 GfuUfgUfcsUfsu AD-53034.1 A-108474.1 CfaAfcAfgCfaUfAfGfuC A-108475.1 uUfuUfaUfuUfgAfcuaUfg faAfaUfaAfaAfL96 CfuGfuUfgsGfsu AD-52976.1 A-108392.1 CfuGfaGfaAfgAfAfCfuA A-108393.1 uUfuAfuAfuGfuAfguuCfu fcAfuAfuAfaAfL96 UfcUfcAfgsUfsu AD-52996.1 A-108336.1 UfgCfuAfuGfuUfAfGfaC A-108337.1 uUfuAfcAfuCfgUfcuaAfc fgAfuGfuAfaAfL96 AfuAfgCfasAfsa AD-53029.1 A-108488.1 AfaCfcCfaCfaGfAfAfaU A-108489.1 aUfaGfaGfaAfaUfuucUfg fuUfcUfcUfaUfL96 UfgGfgUfusCfsu AD-53020.1 A-108438.1 CfuUfcAfaCfaAfAfAfaG A-108439.1 aUfaUfuUfcAfcUfuuuUfg fuGfaAfaUfaUfL96 UfuGfaAfgsUfsa AD-53042.1 A-108414.1 UfcAfcUfuGfaAfCfUfcA A-108415.1 uUfuUfgAfgUfuGfaguUfc faCfuCfaAfaAfL96 AfaGfuGfasCfsa AD-53011.1 A-108482.1 CfaUfaGfuCfaAfAfUfaA A-108483.1 uAfuUfuCfuUfuUfauuUfg faAfgAfaAfuAfL96 AfcUfaUfgsCfsu AD-52957.1 A-108370.1 CfaAfaAfaCfuCfAfAfcA A-108371.1 aUfcAfaAfuAfuGfuugAfg fuAfuUfuGfaUfL96 UfuUfuUfgsAfsa AD-53008.1 A-108434.1 UfaCfuUfcAfaCfAfAfaA A-108435.1 aUfuUfcAfcUfuUfuugUfu faGfuGfaAfaUfL96 GfaAfgUfasGfsa AD-53065.1 A-108496.1 GfaCfcCfaGfcAfAfCfuC A-108497.1 aAfaCfuUfgAfgAfguuGfc fuCfaAfgUfuUfL96 UfgGfgUfcsUfsg AD-53115.1 A-108638.1 UfuGfaAfuGfaAfCfUfgA A-108639.1 aAfuUfuGfcCfuCfaguUfc fgGfcAfaAfuUfL96 AfuUfcAfasAfsg AD-53012.1 A-108404.1 UfaUfaAfaCfuAfCfAfaG A-108405.1 aUfuUfuUfgAfcUfuguAfg fuCfaAfaAfaUfL96 UfuUfaUfasUfsg AD-53004.1 A-108464.1 AfaAfcAfaGfaUfAfAfuA A-108465.1 uUfuGfaUfgCfuAfuuaUfc fgCfaUfcAfaAfL96 UfuGfuUfusUfsu AD-53021.1 A-108454.1 CfaAfcUfaAfcUfAfAfcU A-108455.1 uUfgAfaUfuAfaGfuuaGfu fuAfaUfuCfaAfL96 UfaGfuUfgsCfsu AD-52955.1 A-108338.1 GfcUfaUfgUfuAfGfAfcG A-108339.1 uUfuUfaCfaUfcGfucuAfa faUfgUfaAfaAfL96 CfaUfaGfcsAfsa AD-53119.1 A-108608.1 AfcUfuGfgGfaUfCfAfcA A-108609.1 uUfuUfgCfuUfuGfugaUfc faAfgCfaAfaAfL96 CfcAfaGfusAfsg AD-52990.1 A-108334.1 UfuGfcUfaUfgUfUfAfgA A-108335.1 uUfaCfaUfcGfuCfuaaCfa fcGfaUfgUfaAfL96 UfaGfcAfasAfsu AD-52964.1 A-108388.1 AfaCfuGfaGfaAfGfAfaC A-108389.1 uAfuAfuGfuAfgUfucuUfc fuAfcAfuAfuAfL96 UfcAfgUfusCfsc AD-52973.1 A-108344.1 GfaUfgUfaAfaAfAfUfuU A-108345.1 aUfuGfgCfuAfaAfauuUfu fuAfgCfcAfaUfL96 UfaCfaUfcsGfsu AD-53074.1 A-108546.1 AfcUfcCfaUfaGfUfGfaA A-108547.1 uAfgAfuUfgCfuUfcacUfa fgCfaAfuCfuAfL96 UfgGfaGfusAfsu AD-53026.1 A-108440.1 UfuCfaAfcAfaAfAfAfgU A-108441.1 aAfuAfuUfuCfaCfuuuUfu fgAfaAfuAfuUfL96 GfuUfgAfasGfsu AD-53062.1 A-108542.1 CfuAfgAfgAfaGfAfUfaU A-108543.1 uAfuGfgAfgUfaUfaucUfu faCfuCfcAfuAfL96 CfuCfuAfgsGfsc AD-53114.1 A-108622.1 CfaAfcCfuAfaAfUfGfgU A-108623.1 uUfaUfaUfuUfaCfcauUfu faAfaUfaUfaAfL96 AfgGfuUfgsUfsu AD-53082.1 A-108580.1 GfaAfaUfcAfcGfAfAfaC A-108581.1 uAfuAfgUfuGfgUfuucGfu fcAfaCfuAfuAfL96 GfaUfuUfcsCfsc AD-53035.1 A-108490.1 CfcAfcAfgAfaAfUfUfuC A-108491.1 aAfgAfuAfgAfgAfaauUfu fuCfuAfuCfuUfL96 CfuGfuGfgsGfsu AD-52978.1 A-108330.1 AfaAfuCfaAfgAfUfUfuG A-108331.1 uAfaCfaUfaGfcAfaauCfu fcUfaUfgUfuAfL96 UfgAfuUfusUfsg AD-53084.1 A-108518.1 AfcAfuUfaAfuUfCfAfaC A-108519.1 uAfuUfcGfaUfgUfugaAfu faUfcGfaAfuAfL96 UfaAfuGfusCfsc AD-52972.1 A-108328.1 CfcAfgAfgCfcAfAfAfaU A-108329.1 aAfaUfcUfuGfaUfuuuGfg fcAfaGfaUfuUfL96 CfuCfuGfgsAfsg AD-53002.1 A-108432.1 CfuAfcUfuCfaAfCfAfaA A-108433.1 uUfuCfaCfuUfuUfuguUfg faAfgUfgAfaAfL96 AfaGfuAfgsAfsa AD-53078.1 A-108516.1 GfaCfaUfuAfaUfUfCfaA A-108517.1 aUfuCfgAfuGfuUfgaaUfu fcAfuCfgAfaUfL96 AfaUfgUfcsCfsa AD-53072.1 A-108514.1 GfgAfcAfuUfaAfUfUfcA A-108515.1 uUfcGfaUfgUfuGfaauUfa faCfaUfcGfaAfL96 AfuGfuCfcsAfsu AD-53005.1 A-108480.1 GfcAfuAfgUfcAfAfAfuA A-108481.1 aUfuUfcUfuUfuAfuuuGfa faAfaGfaAfaUfL96 CfuAfuGfcsUfsg AD-53083.1 A-108502.1 CfuCfuCfaAfgUfUfUfuU A-108503.1 uAfgAfcAfuGfaAfaaaCfu fcAfuGfuCfuAfL96 UfgAfgAfgsUfsu AD-53102.1 A-108524.1 AfuCfgAfaUfaGfAfUfgG A-108525.1 uUfuGfuGfaUfcCfaucUfa faUfcAfcAfaAfL96 UfuCfgAfusGfsu AD-53105.1 A-108572.1 AfcAfuUfaUfaUfUfGfaA A-108573.1 aAfaGfaAfuAfuUfcaaUfa fuAfuUfcUfuUfL96 UfaAfuGfusUfsu AD-53090.1 A-108520.1 UfuAfaUfuCfaAfCfAfuC A-108521.1 aUfcUfaUfuCfgAfuguUfg fgAfaUfaGfaUfL96 AfaUfuAfasUfsg AD-53010.1 A-108466.1 GfaUfaAfuAfgCfAfUfcA A-108467.1 aAfgGfuCfuUfuGfaugCfu faAfgAfcCfuUfL96 AfuUfaUfcsUfsu AD-52998.1 A-108368.1 UfgAfcAfuAfuUfUfCfaA A-108369.1 uUfgAfgUfuUfuUfgaaAfu faAfaCfuCfaAfL96 AfuGfuCfasUfsu AD-52992.1 A-108366.1 AfaAfuUfaAfuGfAfCfaU A-108367.1 uUfuGfaAfaUfaUfgucAfu faUfuUfcAfaAfL96 UfaAfuUfusGfsg AD-53068.1 A-108544.1 GfaAfgAfuAfuAfCfUfcC A-108545.1 uUfcAfcUfaUfgGfaguAfu faUfaGfuGfaAfL96 AfuCfuUfcsUfsc AD-53032.1 A-108442.1 AfaUfaUfuUfaGfAfAfgA A-108443.1 uUfaGfuUfgCfuCfuucUfa fgCfaAfcUfaAfL96 AfaUfaUfusUfsc AD-52967.1 A-108342.1 CfgAfuGfuAfaAfAfAfuU A-108343.1 uUfgGfcUfaAfaAfuuuUfu fuUfaGfcCfaAfL96 AfcAfuCfgsUfsc AD-53096.1 A-108522.1 UfuCfaAfcAfuCfGfAfaU A-108523.1 aUfcCfaUfcUfaUfucgAfu faGfaUfgGfaUfL96 GfuUfgAfasUfsu AD-53131.1 A-108612.1 GfuGfuGfgAfgAfAfAfaC A-108613.1 uUfuAfgGfuUfgUfuuuCfu faAfcCfuAfaAfL96 CfcAfcAfcsUfsc AD-52963.1 A-108372.1 UfcAfaCfaUfaUfUfUfgA A-108373.1 aAfgAfcUfgAfuCfaaaUfa fuCfaGfuCfuUfL96 UfgUfuGfasGfsu AD-53089.1 A-108504.1 UfcAfgGfuAfgUfCfCfaU A-108505.1 uAfaUfgUfcCfaUfggaCfu fgGfaCfaUfuAfL96 AfcCfuGfasUfsa AD-53044.1 A-108446.1 UfuUfaGfaAfgAfGfCfaA A-108447.1 uUfaGfuUfaGfuUfgcuCfu fcUfaAfcUfaAfL96 UfcUfaAfasUfsa AD-52988.1 A-108396.1 UfaCfaUfaUfaAfAfCfuA A-108397.1 uUfgAfcUfuGfuAfguuUfa fcAfaGfuCfaAfL96 UfaUfgUfasGfsu AD-53067.1 A-108528.1 GfgAfuCfaCfaAfAfAfcU A-108529.1 uUfcAfuUfgAfaGfuuuUfg fuCfaAfuGfaAfL96 UfgAfuCfcsAfsu AD-53009.1 A-108450.1 AfgAfgCfaAfcUfAfAfcU A-108451.1 aUfuAfaGfuUfaGfuuaGfu faAfcUfuAfaUfL96 UfgCfuCfusUfsc AD-53022.1 A-108470.1 AfcCfaAfcAfgCfAfUfaG A-108471.1 uUfaUfuUfgAfcUfaugCfu fuCfaAfaUfaAfL96 GfuUfgGfusUfsu AD-53016.1 A-108468.1 AfaCfcAfaCfaGfCfAfuA A-108469.1 uAfuUfuGfaCfuAfugcUfg fgUfcAfaAfuAfL96 UfuGfgUfusUfsa AD-53007.1 A-108418.1 GfaAfcUfcAfaCfUfCfaA A-108419.1 uUfcAfaGfuUfuUfgagUfu faAfcUfuGfaAfL96 GfaGfuUfcsAfsa AD-53148.1 A-108634.1 UfaCfuCfuAfuAfAfAfaU A-108635.1 uUfuGfgUfuGfaUfuuuAfu fcAfaCfcAfaAfL96 AfgAfgUfasUfsa AD-53040.1 A-108476.1 CfaGfcAfuAfgUfCfAfaA A-108477.1 uUfcUfuUfuAfuUfugaCfu fuAfaAfaGfaAfL96 AfuGfcUfgsUfsu AD-53041.1 A-108492.1 GfaAfaUfaAfgAfAfAfuG A-108493.1 aUfgUfuUfuAfcAfuuuCfu fuAfaAfaCfaUfL96 UfaUfuUfcsAfsu AD-53039.1 A-108460.1 CfuAfaCfuAfaCfUfUfaA A-108461.1 aUfuUfuGfaAfuUfaagUfu fuUfcAfaAfaUfL96 AfgUfuAfgsUfsu AD-53139.1 A-108646.1 AfuGfaAfcUfgAfGfGfcA A-108647.1 uUfuAfaAfuUfuGfccuCfa faAfuUfuAfaAfL96 GfuUfcAfusUfsc AD-53144.1 A-108648.1 UfgAfaCfuGfaGfGfCfaA A-108649.1 uUfuUfaAfaUfuUfgccUfc faUfuUfaAfaAfL96 AfgUfuCfasUfsu AD-53142.1 A-108616.1 AfaAfcAfaCfcUfAfAfaU A-108617.1 uAfuUfuAfcCfaUfuuaGfg fgGfuAfaAfuAfL96 UfuGfuUfusUfsc AD-53108.1 A-108620.1 AfcAfaCfcUfaAfAfUfgG A-108621.1 uAfuAfuUfuAfcCfauuUfa fuAfaAfuAfuAfL96 GfgUfuGfusUfsu AD-53079.1 A-108532.1 AfaCfgUfgGfgAfGfAfaC A-108533.1 uAfuUfuGfuAfgUfucuCfc fuAfcAfaAfuAfL96 CfaCfgUfusUfsc AD-53133.1 A-108644.1 AfaUfgAfaCfuGfAfGfgC A-108645.1 uUfaAfaUfuUfgCfcucAfg faAfaUfuUfaAfL96 UfuCfaUfusCfsa AD-53104.1 A-108556.1 GfuUfgGfaAfgAfCfUfgG A-108557.1 uUfgUfcUfuUfcCfaguCfu faAfaGfaCfaAfL96 UfcCfaAfcsUfsc AD-53088.1 A-108582.1 UfgGfcAfaUfgUfCfCfcC A-108583.1 aUfuGfcAfuUfgGfggaCfa faAfuGfcAfaUfL96 UfuGfcCfasGfsu AD-53101.1 A-108508.1 GfgUfaGfuCfcAfUfGfgA A-108509.1 aAfuUfaAfuGfuCfcauGfg fcAfuUfaAfuUfL96 AfcUfaCfcsUfsg AD-53000.1 A-108400.1 CfaUfaUfaAfaCfUfAfcA A-108401.1 uUfuUfgAfcUfuGfuagUfu faGfuCfaAfaAfL96 UfaUfaUfgsUfsa AD-53112.1 A-108590.1 AfaUfcCfcGfgAfAfAfaC A-108591.1 aAfaUfcUfuUfgUfuuuCfc faAfaGfaUfuUfL96 GfgGfaUfusGfsc AD-53107.1 A-108604.1 CfuAfcUfuGfgGfAfUfcA A-108605.1 uUfgCfuUfuGfuGfaucCfc fcAfaAfgCfaAfL96 AfaGfuAfgsAfsa AD-53121.1 A-108640.1 UfgAfaUfgAfaCfUfGfaG A-108641.1 aAfaUfuUfgCfcUfcagUfu fgCfaAfaUfuUfL96 CfaUfuCfasAfsa AD-53046.1 A-108478.1 AfgCfaUfaGfuCfAfAfaU A-108479.1 uUfuCfuUfuUfaUfuugAfc faAfaAfgAfaAfL96 UfaUfgCfusGfsu AD-53038.1 A-108444.1 AfuUfuAfgAfaGfAfGfcA A-108445.1 uAfgUfuAfgUfuGfcucUfu faCfuAfaCfuAfL96 CfuAfaAfusAfsu AD-53140.1 A-108662.1 AfgGfcAfaAfuUfUfAfaA A-108663.1 uAfuUfgCfcUfuUfuaaAfu faGfgCfaAfuAfL96 UfuGfcCfusCfsa AD-52987.1 A-108380.1 CfaUfaUfuUfgAfUfCfaG A-108381.1 uAfaAfaAfgAfcUfgauCfa fuCfuUfuUfuAfL96 AfaUfaUfgsUfsu AD-53130.1 A-108596.1 AfaAfaCfaAfaGfAfUfuU A-108597.1 aAfaAfcAfcCfaAfaucUfu fgGfuGfuUfuUfL96 UfgUfuUfusCfsc AD-53106.1 A-108588.1 CfaAfuCfcCfgGfAfAfaA A-108589.1 aAfuCfuUfuGfuUfuucCfg fcAfaAfgAfuUfL96 GfgAfuUfgsCfsa AD-53081.1 A-108564.1 CfaAfcAfaAfcAfUfUfaU A-108565.1 uAfuUfcAfaUfaUfaauGfu faUfuGfaAfuAfL96 UfuGfuUfgsUfsc AD-53118.1 A-108592.1 GfgAfaAfaCfaAfAfGfaU A-108593.1 aAfcAfcCfaAfaUfcuuUfg fuUfgGfuGfuUfL96 UfuUfuCfcsGfsg AD-53136.1 A-108598.1 AfcAfaAfgAfuUfUfGfgU A-108599.1 uAfgAfaAfaCfaCfcaaAfu fgUfuUfuCfuAfL96 CfuUfuGfusUfsu AD-53127.1 A-108642.1 GfaAfuGfaAfcUfGfAfgG A-108643.1 uAfaAfuUfuGfcCfucaGfu fcAfaAfuUfuAfL96 UfcAfuUfcsAfsa AD-53066.1 A-108512.1 CfcAfuGfgAfcAfUfUfaA A-108513.1 aUfgUfuGfaAfuUfaauGfu fuUfcAfaCfaUfL96 CfcAfuGfgsAfsc AD-53013.1 A-108420.1 AfaCfuCfaAfcUfCfAfaA A-108421.1 uUfuCfaAfgUfuUfugaGfu faCfuUfgAfaAfL96 UfgAfgUfusCfsa AD-52991.1 A-108350.1 CfaGfuUfgGfgAfCfAfuG A-108351.1 uUfuAfaGfaCfcAfuguCfc fgUfcUfuAfaAfL96 CfaAfcUfgsAfsa AD-53099.1 A-108570.1 AfaCfaUfuAfuAfUfUfgA A-108571.1 aAfgAfaUfaUfuCfaauAfu faUfaUfuCfuUfL96 AfaUfgUfusUfsg AD-52958.1 A-108386.1 AfcCfaGfuGfaAfAfUfcA A-108387.1 uUfcUfuCfuUfuGfauuUfc faAfgAfaGfaAfL96 AfcUfgGfusUfsu AD-53097.1 A-108538.1 GfuUfgGfgCfcUfAfGfaG A-108539.1 uAfuAfuCfuUfcUfcuaGfg faAfgAfuAfuAfL96 CfcCfaAfcsCfsa AD-52966.1 A-108326.1 CfuCfcAfgAfgCfCfAfaA A-108327.1 aUfcUfuGfaUfuUfuggCfu faUfcAfaGfaUfL96 CfuGfgAfgsAfsu AD-53145.1 A-108664.1 GfgCfaAfaUfuUfAfAfaA A-108665.1 uUfaUfuGfcCfuUfuuaAfa fgGfcAfaUfaAfL96 UfuUfgCfcsUfsc AD-53113.1 A-108606.1 UfaCfuUfgGfgAfUfCfaC A-108607.1 uUfuGfcUfuUfgUfgauCfc faAfaGfcAfaAfL96 CfaAfgUfasGfsa AD-52993.1 A-108382.1 GfaUfcAfgUfcUfUfUfuU A-108383.1 uAfgAfuCfaUfaAfaaaGfa faUfgAfuCfuAfL96 CfuGfaUfcsAfsa AD-53031.1 A-108426.1 GfaAfaGfcCfuCfCfUfaG A-108427.1 uUfuUfuCfuUfcUfaggAfg faAfgAfaAfaAfL96 GfcUfuUfcsAfsa AD-53017.1 A-108484.1 AfgUfcAfaAfuAfAfAfaG A-108485.1 uUfcUfaUfuUfcUfuuuAfu faAfaUfaGfaAfL96 UfuGfaCfusAfsu AD-53143.1 A-108632.1 AfuAfcUfcUfaUfAfAfaA A-108633.1 uUfgGfuUfgAfuUfuuaUfa fuCfaAfcCfaAfL96 GfaGfuAfusAfsa AD-53149.1 A-108650.1 GfaAfcUfgAfgGfCfAfaA A-108651.1 uUfuUfuAfaAfuUfugcCfu fuUfuAfaAfaAfL96 CfaGfuUfcsAfsu AD-53059.1 A-108494.1 AfgAfcCfcAfgCfAfAfcU A-108495.1 aAfcUfuGfaGfaGfuugCfu fcUfcAfaGfuUfL96 GfgGfuCfusGfsa AD-53006.1 A-108402.1 AfuAfuAfaAfcUfAfCfaA A-108403.1 uUfuUfuGfaCfuUfguaGfu fgUfcAfaAfaAfL96 UfuAfuAfusGfsu AD-53025.1 A-108424.1 UfgAfaAfgCfcUfCfCfuA A-108425.1 uUfuUfcUfuCfuAfggaGfg fgAfaGfaAfaAfL96 CfuUfuCfasAfsg AD-53085.1 A-108534.1 GfgGfaGfaAfcUfAfCfaA A-108535.1 aAfcCfaUfaUfuUfguaGfu faUfaUfgGfuUfL96 UfcUfcCfcsAfsc AD-52984.1 A-108332.1 AfgAfuUfuGfcUfAfUfgU A-108333.1 aUfcGfuCfuAfaCfauaGfc fuAfgAfcGfaUfL96 AfaAfuCfusUfsg AD-53023.1 A-108486.1 GfaAfcCfcAfcAfGfAfaA A-108487.1 uAfgAfgAfaAfuUfucuGfu fuUfuCfuCfuAfL96 GfgGfuUfcsUfsu AD-53014.1 A-108436.1 AfcUfuCfaAfcAfAfAfaA A-108437.1 uAfuUfuCfaCfuUfuuuGfu fgUfgAfaAfuAfL96 UfgAfaGfusAfsg AD-53060.1 A-108510.1 AfgUfcCfaUfgGfAfCfaU A-108511.1 uUfgAfaUfuAfaUfgucCfa fuAfaUfuCfaAfL96 UfgGfaCfusAfsc AD-53110.1 A-108652.1 AfaCfuGfaGfgCfAfAfaU A-108653.1 uCfuUfuUfaAfaUfuugCfc fuUfaAfaAfgAfL96 UfcAfgUfusCfsa AD-52980.1 A-108362.1 GfgGfcCfaAfaUfUfAfaU A-108363.1 aAfuAfuGfuCfaUfuaaUfu fgAfcAfuAfuUfL96 UfgGfcCfcsUfsu AD-53109.1 A-108636.1 AfuCfcAfuCfcAfAfCfaG A-108637.1 uUfcUfgAfaUfcUfguuGfg faUfuCfaGfaAfL96 AfuGfgAfusCfsa AD-53141.1 A-108600.1 AfaGfaUfuUfgGfUfGfuU A-108601.1 aAfgUfaGfaAfaAfcacCfa fuUfcUfaCfuUfL96 AfaUfcUfusUfsg AD-53126.1 A-108626.1 GfuCfuCfaAfaAfUfGfgA A-108627.1 uAfuAfaCfcUfuCfcauUfu faGfgUfuAfuAfL96 UfgAfgAfcsUfsu AD-53116.1 A-108654.1 AfcUfgAfgGfcAfAfAfuU A-108655.1 uCfcUfuUfuAfaAfuuuGfc fuAfaAfaGfgAfL96 CfuCfaGfusUfsc AD-52997.1 A-108352.1 GfgGfaCfaUfgGfUfCfuU A-108353.1 aAfgUfcUfuUfaAfgacCfa faAfaGfaCfuUfL96 UfgUfcCfcsAfsa AD-53120.1 A-108624.1 AfuGfgUfaAfaUfAfUfaA A-108625.1 uUfgGfuUfuGfuUfauaUfu fcAfaAfcCfaAfL96 UfaCfcAfusUfsu AD-53070.1 A-108576.1 GfgGfaAfaUfcAfCfGfaA A-108577.1 uAfgUfuGfgUfuUfcguGfa faCfcAfaCfuAfL96 UfuUfcCfcsAfsa AD-53028.1 A-108472.1 CfcAfaCfaGfcAfUfAfgU A-108473.1 uUfuAfuUfuGfaCfuauGfc fcAfaAfuAfaAfL96 UfgUfuGfgsUfsu AD-53146.1 A-108602.1 UfuUfuCfuAfcUfUfGfgG A-108603.1 uUfuGfuGfaUfcCfcaaGfu faUfcAfcAfaAfL96 AfgAfaAfasCfsa AD-52982.1 A-108394.1 AfgAfaCfuAfcAfUfAfuA A-108395.1 uUfgUfaGfuUfuAfuauGfu faAfcUfaCfaAfL96 AfgUfuCfusUfsc AD-53111.1 A-108668.1 AfgAfgUfaUfgUfGfUfaA A-108669.1 aCfaGfaUfuUfuUfacaCfa faAfaUfcUfgUfL96 UfaCfuCfusGfsu AD-53045.1 A-108462.1 AfaAfaCfaAfgAfUfAfaU A-108463.1 uUfgAfuGfcUfaUfuauCfu faGfcAfuCfaAfL96 UfgUfuUfusUfsc AD-53123.1 A-108672.1 AfgUfaUfgUfgUfAfAfaA A-108673.1 uUfaCfaGfaUfuUfuuaCfa faUfcUfgUfaAfL96 CfaUfaCfusCfsu AD-53018.1 A-108406.1 AfgUfcAfaAfaAfUfGfaA A-108407.1 uUfuAfcCfuCfuUfcauUfu fgAfgGfuAfaAfL96 UfuGfaCfusUfsg AD-52956.1 A-108354.1 GfgAfcAfuGfgUfCfUfuA A-108355.1 aAfaGfuCfuUfuAfagaCfc faAfgAfcUfuUfL96 AfuGfuCfcsCfsa AD-53134.1 A-108660.1 GfaGfgCfaAfaUfUfUfaA A-108661.1 aUfuGfcCfuUfuUfaaaUfu faAfgGfcAfaUfL96 UfgCfcUfcsAfsg AD-52968.1 A-108358.1 GfuCfuUfaAfaGfAfCfuU A-108359.1 uAfuGfgAfcAfaAfgucUfu fuGfuCfcAfuAfL96 UfaAfgAfcsCfsa AD-53122.1 A-108656.1 CfuGfaGfgCfaAfAfUfuU A-108657.1 uGfcCfuUfuUfaAfauuUfg faAfaAfgGfcAfL96 CfcUfcAfgsUfsu AD-53100.1 A-108586.1 GfcAfaUfcCfcGfGfAfaA A-108587.1 aUfcUfuUfgUfuUfuccGfg faCfaAfaGfaUfL96 GfaUfuGfcsAfsu AD-53128.1 A-108658.1 UfgAfgGfcAfaAfUfUfuA A-108659.1 uUfgCfcUfuUfuAfaauUfu faAfaGfgCfaAfL96 GfcCfuCfasGfsu AD-53043.1 A-108430.1 UfcUfaCfuUfcAfAfCfaA A-108431.1 uUfcAfcUfuUfuUfguuGfa faAfaGfuGfaAfL96 AfgUfaGfasAfsu AD-53135.1 A-108676.1 UfaUfgUfgUfaAfAfAfaU A-108677.1 uAfuUfaCfaGfaUfuuuUfa fcUfgUfaAfuAfL96 CfaCfaUfasCfsu AD-53094.1 A-108584.1 AfaUfgCfaAfuCfCfCfgG A-108585.1 uUfuGfuUfuUfcCfgggAfu faAfaAfcAfaAfL96 UfgCfaUfusGfsg AD-53019.1 A-108422.1 CfuUfgAfaAfgCfCfUfcC A-108423.1 uUfcUfuCfuAfgGfaggCfu fuAfgAfaGfaAfL96 UfuCfaAfgsUfsu AD-53129.1 A-108674.1 GfuAfuGfuGfuAfAfAfaA A-108675.1 aUfuAfcAfgAfuUfuuuAfc fuCfuGfuAfaUfL96 AfcAfuAfcsUfsc AD-53150.1 A-108666.1 CfaGfaGfuAfuGfUfGfuA A-108667.1 aAfgAfuUfuUfuAfcacAfu faAfaAfuCfuUfL96 AfcUfcUfgsUfsg AD-53117.1 A-108670.1 GfaGfuAfuGfuGfUfAfaA A-108671.1 uAfcAfgAfuUfuUfuacAfc faAfuCfuGfuAfL96 AfuAfcUfcsUfsg AD-52985.1 A-108348.1 UfcAfgUfuGfgGfAfCfaU A-108349.1 uUfaAfgAfcCfaUfgucCfc fgGfuCfuUfaAfL96 AfaCfuGfasAfsg AD-52962.1 A-108356.1 GfgUfcUfuAfaAfGfAfcU A-108357.1 aUfgGfaCfaAfaGfucuUfu fuUfgUfcCfaUfL96 AfaGfaCfcsAfsu AD-52974.1 A-108360.1 UfcUfuAfaAfgAfCfUfuU A-108361.1 uUfaUfgGfaCfaAfaguCfu fgUfcCfaUfaAfL96 UfuAfaGfasCfsc AD-52979.1 A-108346.1 UfuCfaGfuUfgGfGfAfcA A-108347.1 uAfaGfaCfcAfuGfuccCfa fuGfgUfcUfuAfL96 AfcUfgAfasGfsg Lowercase nucleotides (a, u, g, c) are 2′-O-methyl nucleotides; Nf (e.g., Af) is a 2′-fluoro nucleotide; s is a phosphothiorate linkage; L96 indicates a GalNAc ligand.

TABLE 9 Unmodified Sense and antisense strand sequences of ANGPTL3 dsRNAs without GalNal conjugation These sequences are the same as the sequences listed in Table 7 except that they do not contain GalNal conjugation. Sense Sequence Antisense Sequence (SEQ ID NOS 1004-1184, (SEQ ID NOS 1185-1365, Duplex Sense respectively, in Antisense respectively, in order Position in Name OligoName order of appearance) OligoName of appearance) NM_014495.2 AD-52637.1 A-108817.1 UCACAAUUAAGCUCCUUCUUU A-108307.2 AAAGAAGGAGCUUAAUUGUGAAC   54-76 AD-52638.1 A-108825.1 UUAUUGUUCCUCUAGUUAUUU A-108323.2 AAAUAACUAGAGGAACAAUAAAA   75-97 AD-52639.1 A-108833.1 GCUAUGUUAGACGAUGUAAAA A-108339.2 UUUUACAUCGUCUAACAUAGCAA  161-183 AD-52640.1 A-108841.1 GGACAUGGUCUUAAAGACUUU A-108355.2 AAAGUCUUUAAGACCAUGUCCCA  209-231 AD-52641.1 A-108849.1 CAAAAACUCAACAUAUUUGAU A-108371.2 AUCAAAUAUGUUGAGUUUUUGAA  266-288 AD-52642.1 A-108857.1 ACCAGUGAAAUCAAAGAAGAA A-108387.2 UUCUUCUUUGAUUUCACUGGUUU  314-336 AD-52643.1 A-108818.1 CACAAUUAAGCUCCUUCUUUU A-108309.2 AAAAGAAGGAGCUUAAUUGUGAA   55-77 AD-52645.1 A-108834.1 CUAUGUUAGACGAUGUAAAAA A-108341.2 UUUUUACAUCGUCUAACAUAGCA  162-184 AD-52647.1 A-108850.1 UCAACAUAUUUGAUCAGUCUU A-108373.2 AAGACUGAUCAAAUAUGUUGAGU  273-295 AD-52648.1 A-108858.1 AACUGAGAAGAACUACAUAUA A-108389.2 UAUAUGUAGUUCUUCUCAGUUCC  342-364 AD-52649.1 A-108819.1 ACAAUUAAGCUCCUUCUUUUU A-108311.2 AAAAAGAAGGAGCUUAAUUGUGA   56-78 AD-52650.1 A-108827.1 CUCCAGAGCCAAAAUCAAGAU A-108327.2 AUCUUGAUUUUGGCUCUGGAGAU  138-160 AD-52651.1 A-108835.1 CGAUGUAAAAAUUUUAGCCAA A-108343.2 UUGGCUAAAAUUUUUACAUCGUC  172-194 AD-52652.1 A-108843.1 GUCUUAAAGACUUUGUCCAUA A-108359.2 UAUGGACAAAGUCUUUAAGACCA  216-238 AD-52653.1 A-108851.1 CAACAUAUUUGAUCAGUCUUU A-108375.2 AAAGACUGAUCAAAUAUGUUGAG  274-296 AD-52654.1 A-108859.1 ACUGAGAAGAACUACAUAUAA A-108391.2 UUAUAUGUAGUUCUUCUCAGUUC  343-365 AD-52656.1 A-108828.1 CCAGAGCCAAAAUCAAGAUUU A-108329.2 AAAUCUUGAUUUUGGCUCUGGAG  140-162 AD-52657.1 A-108836.1 GAUGUAAAAAUUUUAGCCAAU A-108345.2 AUUGGCUAAAAUUUUUACAUCGU  173-195 AD-52658.1 A-108844.1 UCUUAAAGACUUUGUCCAUAA A-108361.2 UUAUGGACAAAGUCUUUAAGACC  217-239 AD-52659.1 A-108852.1 AACAUAUUUGAUCAGUCUUUU A-108377.2 AAAAGACUGAUCAAAUAUGUUGA  275-297 AD-52660.1 A-108860.1 CUGAGAAGAACUACAUAUAAA A-108393.2 UUUAUAUGUAGUUCUUCUCAGUU  344-366 AD-52661.1 A-108821.1 AAUUAAGCUCCUUCUUUUUAU A-108315.2 AUAAAAAGAAGGAGCUUAAUUGU   58-80 AD-52662.1 A-108829.1 AAAUCAAGAUUUGCUAUGUUA A-108331.2 UAACAUAGCAAAUCUUGAUUUUG  149-171 AD-52663.1 A-108837.1 UUCAGUUGGGACAUGGUCUUA A-108347.2 UAAGACCAUGUCCCAACUGAAGG  201-223 AD-52664.1 A-108845.1 GGGCCAAAUUAAUGACAUAUU A-108363.2 AAUAUGUCAUUAAUUUGGCCCUU  244-266 AD-52665.1 A-108853.1 ACAUAUUUGAUCAGUCUUUUU A-108379.2 AAAAAGACUGAUCAAAUAUGUUG  276-298 AD-52666.1 A-108861.1 AGAACUACAUAUAAACUACAA A-108395.2 UUGUAGUUUAUAUGUAGUUCUUC  350-372 AD-52667.1 A-108822.1 AUUAAGCUCCUUCUUUUUAUU A-108317.2 AAUAAAAAGAAGGAGCUUAAUUG   59-81 AD-52668.1 A-108830.1 AGAUUUGCUAUGUUAGACGAU A-108333.2 AUCGUCUAACAUAGCAAAUCUUG  155-177 AD-52669.1 A-108838.1 UCAGUUGGGACAUGGUCUUAA A-108349.2 UUAAGACCAUGUCCCAACUGAAG  202-224 AD-52670.1 A-108846.1 GGCCAAAUUAAUGACAUAUUU A-108365.2 AAAUAUGUCAUUAAUUUGGCCCU  245-267 AD-52671.1 A-108854.1 CAUAUUUGAUCAGUCUUUUUA A-108381.2 UAAAAAGACUGAUCAAAUAUGUU  277-299 AD-52672.1 A-108862.1 UACAUAUAAACUACAAGUCAA A-108397.2 UUGACUUGUAGUUUAUAUGUAGU  355-377 AD-52673.1 A-108823.1 UUUUAUUGUUCCUCUAGUUAU A-108319.2 AUAACUAGAGGAACAAUAAAAAG   73-95 AD-52674.1 A-108831.1 UUGCUAUGUUAGACGAUGUAA A-108335.2 UUACAUCGUCUAACAUAGCAAAU  159-181 AD-52675.1 A-108839.1 CAGUUGGGACAUGGUCUUAAA A-108351.2 UUUAAGACCAUGUCCCAACUGAA  203-225 AD-52676.1 A-108847.1 AAAUUAAUGACAUAUUUCAAA A-108367.2 UUUGAAAUAUGUCAUUAAUUUGG  249-271 AD-52677.1 A-108855.1 GAUCAGUCUUUUUAUGAUCUA A-108383.2 UAGAUCAUAAAAAGACUGAUCAA  284-306 AD-52678.1 A-108863.1 ACAUAUAAACUACAAGUCAAA A-108399.2 UUUGACUUGUAGUUUAUAUGUAG  356-378 AD-52679.1 A-108824.1 UUUAUUGUUCCUCUAGUUAUU A-108321.2 AAUAACUAGAGGAACAAUAAAAA   74-96 AD-52680.1 A-108832.1 UGCUAUGUUAGACGAUGUAAA A-108337.2 UUUACAUCGUCUAACAUAGCAAA  160-182 AD-52681.1 A-108840.1 GGGACAUGGUCUUAAAGACUU A-108353.2 AAGUCUUUAAGACCAUGUCCCAA  208-230 AD-52682.1 A-108848.1 UGACAUAUUUCAAAAACUCAA A-108369.2 UUGAGUUUUUGAAAUAUGUCAUU  256-278 AD-52683.1 A-108856.1 AUCAGUCUUUUUAUGAUCUAU A-108385.2 AUAGAUCAUAAAAAGACUGAUCA  285-307 AD-52684.1 A-108864.1 CAUAUAAACUACAAGUCAAAA A-108401.2 UUUUGACUUGUAGUUUAUAUGUA  357-379 AD-52685.1 A-108872.1 CUUGAACUCAACUCAAAACUU A-108417.2 AAGUUUUGAGUUGAGUUCAAGUG  401-423 AD-52686.1 A-108880.1 CUACUUCAACAAAAAGUGAAA A-108433.2 UUUCACUUUUUGUUGAAGUAGAA  446-468 AD-52687.1 A-108888.1 AAGAGCAACUAACUAACUUAA A-108449.2 UUAAGUUAGUUAGUUGCUCUUCU  474-496 AD-52688.1 A-108896.1 AAACAAGAUAAUAGCAUCAAA A-108465.2 UUUGAUGCUAUUAUCUUGUUUUU  557-579 AD-52689.1 A-108904.1 GCAUAGUCAAAUAAAAGAAAU A-108481.2 AUUUCUUUUAUUUGACUAUGCUG  625-647 AD-52690.1 A-108865.1 AUAUAAACUACAAGUCAAAAA A-108403.2 UUUUUGACUUGUAGUUUAUAUGU  358-380 AD-52691.1 A-108873.1 GAACUCAACUCAAAACUUGAA A-108419.2 UUCAAGUUUUGAGUUGAGUUCAA  404-426 AD-52692.1 A-108881.1 UACUUCAACAAAAAGUGAAAU A-108435.2 AUUUCACUUUUUGUUGAAGUAGA  447-469 AD-52693.1 A-108889.1 AGAGCAACUAACUAACUUAAU A-108451.2 AUUAAGUUAGUUAGUUGCUCUUC  475-497 AD-52694.1 A-108897.1 GAUAAUAGCAUCAAAGACCUU A-108467.2 AAGGUCUUUGAUGCUAUUAUCUU  563-585 AD-52695.1 A-108905.1 CAUAGUCAAAUAAAAGAAAUA A-108483.2 UAUUUCUUUUAUUUGACUAUGCU  626-648 AD-52696.1 A-108866.1 UAUAAACUACAAGUCAAAAAU A-108405.2 AUUUUUGACUUGUAGUUUAUAUG  359-381 AD-52697.1 A-108874.1 AACUCAACUCAAAACUUGAAA A-108421.2 UUUCAAGUUUUGAGUUGAGUUCA  405-427 AD-52698.1 A-108882.1 ACUUCAACAAAAAGUGAAAUA A-108437.2 UAUUUCACUUUUUGUUGAAGUAG  448-470 AD-52699.1 A-108890.1 GAGCAACUAACUAACUUAAUU A-108453.2 AAUUAAGUUAGUUAGUUGCUCUU  476-498 AD-52700.1 A-108898.1 AACCAACAGCAUAGUCAAAUA A-108469.2 UAUUUGACUAUGCUGUUGGUUUA  617-639 AD-52701.1 A-108906.1 AGUCAAAUAAAAGAAAUAGAA A-108485.2 UUCUAUUUCUUUUAUUUGACUAU  629-651 AD-52702.1 A-108867.1 AGUCAAAAAUGAAGAGGUAAA A-108407.2 UUUACCUCUUCAUUUUUGACUUG  370-392 AD-52703.1 A-108875.1 CUUGAAAGCCUCCUAGAAGAA A-108423.2 UUCUUCUAGGAGGCUUUCAAGUU  419-441 AD-52704.1 A-108883.1 CUUCAACAAAAAGUGAAAUAU A-108439.2 AUAUUUCACUUUUUGUUGAAGUA  449-471 AD-52705.1 A-108891.1 CAACUAACUAACUUAAUUCAA A-108455.2 UUGAAUUAAGUUAGUUAGUUGCU  479-501 AD-52706.1 A-108899.1 ACCAACAGCAUAGUCAAAUAA A-108471.2 UUAUUUGACUAUGCUGUUGGUUU  618-640 AD-52707.1 A-108907.1 GAACCCACAGAAAUUUCUCUA A-108487.2 UAGAGAAAUUUCUGUGGGUUCUU  677-699 AD-52708.1 A-108868.1 GAAUAUGUCACUUGAACUCAA A-108409.2 UUGAGUUCAAGUGACAUAUUCUU  391-413 AD-52709.1 A-108876.1 UGAAAGCCUCCUAGAAGAAAA A-108425.2 UUUUCUUCUAGGAGGCUUUCAAG  421-443 AD-52710.1 A-108884.1 UUCAACAAAAAGUGAAAUAUU A-108441.2 AAUAUUUCACUUUUUGUUGAAGU  450-472 AD-52711.1 A-108892.1 AACUAACUAACUUAAUUCAAA A-108457.2 UUUGAAUUAAGUUAGUUAGUUGC  480-502 AD-52712.1 A-108900.1 CCAACAGCAUAGUCAAAUAAA A-108473.2 UUUAUUUGACUAUGCUGUUGGUU  619-641 AD-52713.1 A-108908.1 AACCCACAGAAAUUUCUCUAU A-108489.2 AUAGAGAAAUUUCUGUGGGUUCU  678-700 AD-52714.1 A-108869.1 UGUCACUUGAACUCAACUCAA A-108411.2 UUGAGUUGAGUUCAAGUGACAUA  396-418 AD-52715.1 A-108877.1 GAAAGCCUCCUAGAAGAAAAA A-108427.2 UUUUUCUUCUAGGAGGCUUUCAA  422-444 AD-52716.1 A-108885.1 AAUAUUUAGAAGAGCAACUAA A-108443.2 UUAGUUGCUCUUCUAAAUAUUUC  465-487 AD-52717.1 A-108893.1 ACUAACUAACUUAAUUCAAAA A-108459.2 UUUUGAAUUAAGUUAGUUAGUUG  481-503 AD-52718.1 A-108901.1 CAACAGCAUAGUCAAAUAAAA A-108475.2 UUUUAUUUGACUAUGCUGUUGGU  620-642 AD-52719.1 A-108909.1 CCACAGAAAUUUCUCUAUCUU A-108491.2 AAGAUAGAGAAAUUUCUGUGGGU  681-703 AD-52720.1 A-108870.1 GUCACUUGAACUCAACUCAAA A-108413.2 UUUGAGUUGAGUUCAAGUGACAU  397-419 AD-52721.1 A-108878.1 CUCCUAGAAGAAAAAAUUCUA A-108429.2 UAGAAUUUUUUCUUCUAGGAGGC  428-450 AD-52722.1 A-108886.1 AUUUAGAAGAGCAACUAACUA A-108445.2 UAGUUAGUUGCUCUUCUAAAUAU  468-490 AD-52723.1 A-108894.1 CUAACUAACUUAAUUCAAAAU A-108461.2 AUUUUGAAUUAAGUUAGUUAGUU  482-504 AD-52724.1 A-108902.1 CAGCAUAGUCAAAUAAAAGAA A-108477.2 UUCUUUUAUUUGACUAUGCUGUU  623-645 AD-52725.1 A-108910.1 GAAAUAAGAAAUGUAAAACAU A-108493.2 AUGUUUUACAUUUCUUAUUUCAU  746-768 AD-52726.1 A-108871.1 UCACUUGAACUCAACUCAAAA A-108415.2 UUUUGAGUUGAGUUCAAGUGACA  398-420 AD-52727.1 A-108879.1 UCUACUUCAACAAAAAGUGAA A-108431.2 UUCACUUUUUGUUGAAGUAGAAU  445-467 AD-52728.1 A-108887.1 UUUAGAAGAGCAACUAACUAA A-108447.2 UUAGUUAGUUGCUCUUCUAAAUA  469-491 AD-52729.1 A-108895.1 AAAACAAGAUAAUAGCAUCAA A-108463.2 UUGAUGCUAUUAUCUUGUUUUUC  556-578 AD-52730.1 A-108903.1 AGCAUAGUCAAAUAAAAGAAA A-108479.2 UUUCUUUUAUUUGACUAUGCUGU  624-646 AD-52731.1 A-108958.1 AGACCCAGCAACUCUCAAGUU A-108495.2 AACUUGAGAGUUGCUGGGUCUGA  836-858 AD-52732.1 A-108966.1 AGUCCAUGGACAUUAAUUCAA A-108511.2 UUGAAUUAAUGUCCAUGGACUAC  887-909 AD-52733.1 A-108974.1 GAUGGAUCACAAAACUUCAAU A-108527.2 AUUGAAGUUUUGUGAUCCAUCUA  917-939 AD-52734.1 A-108982.1 CUAGAGAAGAUAUACUCCAUA A-108543.2 UAUGGAGUAUAUCUUCUCUAGGC  998-1020 AD-52735.1 A-108990.1 AAAGACAACAAACAUUAUAUU A-108559.2 AAUAUAAUGUUUGUUGUCUUUCC 1064-1086 AD-52736.1 A-108998.1 CAUUAUAUUGAAUAUUCUUUU A-108575.2 AAAAGAAUAUUCAAUAUAAUGUU 1076-1098 AD-52737.1 A-108959.1 GACCCAGCAACUCUCAAGUUU A-108497.2 AAACUUGAGAGUUGCUGGGUCUG  837-859 AD-52739.1 A-108975.1 GGAUCACAAAACUUCAAUGAA A-108529.2 UUCAUUGAAGUUUUGUGAUCCAU  920-942 AD-52740.1 A-108983.1 GAAGAUAUACUCCAUAGUGAA A-108545.2 UUCACUAUGGAGUAUAUCUUCUC 1003-1025 AD-52741.1 A-108991.1 GACAACAAACAUUAUAUUGAA A-108561.2 UUCAAUAUAAUGUUUGUUGUCUU 1067-1089 AD-52742.1 A-108999.1 GGGAAAUCACGAAACCAACUA A-108577.2 UAGUUGGUUUCGUGAUUUCCCAA 1102-1124 AD-52743.1 A-108960.1 ACCCAGCAACUCUCAAGUUUU A-108499.2 AAAACUUGAGAGUUGCUGGGUCU  838-860 AD-52744.1 A-108968.1 GGACAUUAAUUCAACAUCGAA A-108515.2 UUCGAUGUUGAAUUAAUGUCCAU  894-916 AD-52745.1 A-108976.1 GAUCACAAAACUUCAAUGAAA A-108531.2 UUUCAUUGAAGUUUUGUGAUCCA  921-943 AD-52746.1 A-108984.1 ACUCCAUAGUGAAGCAAUCUA A-108547.2 UAGAUUGCUUCACUAUGGAGUAU 1011-1033 AD-52747.1 A-108992.1 ACAACAAACAUUAUAUUGAAU A-108563.2 AUUCAAUAUAAUGUUUGUUGUCU 1068-1090 AD-52748.1 A-109000.1 GGAAAUCACGAAACCAACUAU A-108579.2 AUAGUUGGUUUCGUGAUUUCCCA 1103-1125 AD-52749.1 A-108961.1 CCCAGCAACUCUCAAGUUUUU A-108501.2 AAAAACUUGAGAGUUGCUGGGUC  839-861 AD-52750.1 A-108969.1 GACAUUAAUUCAACAUCGAAU A-108517.2 AUUCGAUGUUGAAUUAAUGUCCA  895-917 AD-52751.1 A-108977.1 AACGUGGGAGAACUACAAAUA A-108533.2 UAUUUGUAGUUCUCCCACGUUUC  940-962 AD-52752.1 A-108985.1 CUCCAUAGUGAAGCAAUCUAA A-108549.2 UUAGAUUGCUUCACUAUGGAGUA 1012-1034 AD-52753.1 A-108993.1 CAACAAACAUUAUAUUGAAUA A-108565.2 UAUUCAAUAUAAUGUUUGUUGUC 1069-1091 AD-52754.1 A-109001.1 GAAAUCACGAAACCAACUAUA A-108581.2 UAUAGUUGGUUUCGUGAUUUCCC 1104-1126 AD-52755.1 A-108962.1 CUCUCAAGUUUUUCAUGUCUA A-108503.2 UAGACAUGAAAAACUUGAGAGUU  847-869 AD-52756.1 A-108970.1 ACAUUAAUUCAACAUCGAAUA A-108519.2 UAUUCGAUGUUGAAUUAAUGUCC  896-918 AD-52757.1 A-108978.1 GGGAGAACUACAAAUAUGGUU A-108535.2 AACCAUAUUUGUAGUUCUCCCAC  945-967 AD-52758.1 A-108986.1 UCCAUAGUGAAGCAAUCUAAU A-108551.2 AUUAGAUUGCUUCACUAUGGAGU 1013-1035 AD-52759.1 A-108994.1 AACAAACAUUAUAUUGAAUAU A-108567.2 AUAUUCAAUAUAAUGUUUGUUGU 1070-1092 AD-52760.1 A-109002.1 UGGCAAUGUCCCCAAUGCAAU A-108583.2 AUUGCAUUGGGGACAUUGCCAGU 1147-1169 AD-52761.1 A-108963.1 UCAGGUAGUCCAUGGACAUUA A-108505.2 UAAUGUCCAUGGACUACCUGAUA  881-903 AD-52762.1 A-108971.1 UUAAUUCAACAUCGAAUAGAU A-108521.2 AUCUAUUCGAUGUUGAAUUAAUG  899-921 AD-52763.1 A-108979.1 GGAGAACUACAAAUAUGGUUU A-108537.2 AAACCAUAUUUGUAGUUCUCCCA  946-968 AD-52764.1 A-108987.1 CCAUAGUGAAGCAAUCUAAUU A-108553.2 AAUUAGAUUGCUUCACUAUGGAG 1014-1036 AD-52765.1 A-108995.1 ACAAACAUUAUAUUGAAUAUU A-108569.2 AAUAUUCAAUAUAAUGUUUGUUG 1071-1093 AD-52766.1 A-109003.1 AAUGCAAUCCCGGAAAACAAA A-108585.2 UUUGUUUUCCGGGAUUGCAUUGG 1160-1182 AD-52767.1 A-108964.1 CAGGUAGUCCAUGGACAUUAA A-108507.2 UUAAUGUCCAUGGACUACCUGAU  882-904 AD-52768.1 A-108972.1 UUCAACAUCGAAUAGAUGGAU A-108523.2 AUCCAUCUAUUCGAUGUUGAAUU  903-925 AD-52769.1 A-108980.1 GUUGGGCCUAGAGAAGAUAUA A-108539.2 UAUAUCUUCUCUAGGCCCAACCA  991-1013 AD-52770.1 A-108988.1 CAUAGUGAAGCAAUCUAAUUA A-108555.2 UAAUUAGAUUGCUUCACUAUGGA 1015-1037 AD-52771.1 A-108996.1 AACAUUAUAUUGAAUAUUCUU A-108571.2 AAGAAUAUUCAAUAUAAUGUUUG 1074-1096 AD-52772.1 A-109004.1 GCAAUCCCGGAAAACAAAGAU A-108587.2 AUCUUUGUUUUCCGGGAUUGCAU 1163-1185 AD-52773.1 A-108965.1 GGUAGUCCAUGGACAUUAAUU A-108509.2 AAUUAAUGUCCAUGGACUACCUG  884-906 AD-52774.1 A-108973.1 AUCGAAUAGAUGGAUCACAAA A-108525.2 UUUGUGAUCCAUCUAUUCGAUGU  909-931 AD-52775.1 A-108981.1 CCUAGAGAAGAUAUACUCCAU A-108541.2 AUGGAGUAUAUCUUCUCUAGGCC  997-1019 AD-52776.1 A-108989.1 GUUGGAAGACUGGAAAGACAA A-108557.2 UUGUCUUUCCAGUCUUCCAACUC 1051-1073 AD-52777.1 A-108997.1 ACAUUAUAUUGAAUAUUCUUU A-108573.2 AAAGAAUAUUCAAUAUAAUGUUU 1075-1097 AD-52778.1 A-109005.1 CAAUCCCGGAAAACAAAGAUU A-108589.2 AAUCUUUGUUUUCCGGGAUUGCA 1164-1186 AD-52779.1 A-109013.1 CUACUUGGGAUCACAAAGCAA A-108605.2 UUGCUUUGUGAUCCCAAGUAGAA 1194-1216 AD-52780.1 A-109021.1 ACAACCUAAAUGGUAAAUAUA A-108621.2 UAUAUUUACCAUUUAGGUUGUUU 1281-1303 AD-52781.1 A-109029.1 AUCCAUCCAACAGAUUCAGAA A-108637.2 UUCUGAAUCUGUUGGAUGGAUCA 1400-1422 AD-52782.1 A-109037.1 AACUGAGGCAAAUUUAAAAGA A-108653.2 UCUUUUAAAUUUGCCUCAGUUCA 1432-1454_G21A AD-52783.1 A-109045.1 AGAGUAUGUGUAAAAAUCUGU A-108669.2 ACAGAUUUUUACACAUACUCUGU 1913-1935 AD-52784.1 A-109006.1 AAUCCCGGAAAACAAAGAUUU A-108591.2 AAAUCUUUGUUUUCCGGGAUUGC 1165-1187 AD-52785.1 A-109014.1 UACUUGGGAUCACAAAGCAAA A-108607.2 UUUGCUUUGUGAUCCCAAGUAGA 1195-1217 AD-52786.1 A-109022.1 CAACCUAAAUGGUAAAUAUAA A-108623.2 UUAUAUUUACCAUUUAGGUUGUU 1282-1304 AD-52787.1 A-109030.1 UUGAAUGAACUGAGGCAAAUU A-108639.2 AAUUUGCCUCAGUUCAUUCAAAG 1425-1447 AD-52788.1 A-109038.1 ACUGAGGCAAAUUUAAAAGGA A-108655.2 UCCUUUUAAAUUUGCCUCAGUUC 1433-1455_C21A AD-52789.1 A-109046.1 GAGUAUGUGUAAAAAUCUGUA A-108671.2 UACAGAUUUUUACACAUACUCUG 1914-1936 AD-52791.1 A-109015.1 ACUUGGGAUCACAAAGCAAAA A-108609.2 UUUUGCUUUGUGAUCCCAAGUAG 1196-1218 AD-52792.1 A-109023.1 AUGGUAAAUAUAACAAACCAA A-108625.2 UUGGUUUGUUAUAUUUACCAUUU 1290-1312 AD-52793.1 A-109031.1 UGAAUGAACUGAGGCAAAUUU A-108641.2 AAAUUUGCCUCAGUUCAUUCAAA 1426-1448 AD-52794.1 A-109039.1 CUGAGGCAAAUUUAAAAGGCA A-108657.2 UGCCUUUUAAAUUUGCCUCAGUU 1434-1456 AD-52795.1 A-109047.1 AGUAUGUGUAAAAAUCUGUAA A-108673.2 UUACAGAUUUUUACACAUACUCU 1915-1937 AD-52796.1 A-109008.1 GAAAACAAAGAUUUGGUGUUU A-108595.2 AAACACCAAAUCUUUGUUUUCCG 1172-1194 AD-52797.1 A-109016.1 AGUGUGGAGAAAACAACCUAA A-108611.2 UUAGGUUGUUUUCUCCACACUCA 1269-1291 AD-52798.1 A-109024.1 GUCUCAAAAUGGAAGGUUAUA A-108627.2 UAUAACCUUCCAUUUUGAGACUU 1354-1376 AD-52799.1 A-109032.1 GAAUGAACUGAGGCAAAUUUA A-108643.2 UAAAUUUGCCUCAGUUCAUUCAA 1427-1449 AD-52800.1 A-109040.1 UGAGGCAAAUUUAAAAGGCAA A-108659.2 UUGCCUUUUAAAUUUGCCUCAGU 1435-1457 AD-52801.1 A-109048.1 GUAUGUGUAAAAAUCUGUAAU A-108675.2 AUUACAGAUUUUUACACAUACUC 1916-1938 AD-52802.1 A-109009.1 AAAACAAAGAUUUGGUGUUUU A-108597.2 AAAACACCAAAUCUUUGUUUUCC 1173-1195 AD-52803.1 A-109017.1 GUGUGGAGAAAACAACCUAAA A-108613.2 UUUAGGUUGUUUUCUCCACACUC 1270-1292 AD-52804.1 A-109025.1 AUGGAAGGUUAUACUCUAUAA A-108629.2 UUAUAGAGUAUAACCUUCCAUUU 1362-1384 AD-52805.1 A-109033.1 AAUGAACUGAGGCAAAUUUAA A-108645.2 UUAAAUUUGCCUCAGUUCAUUCA 1428-1450 AD-52806.1 A-109041.1 GAGGCAAAUUUAAAAGGCAAU A-108661.2 AUUGCCUUUUAAAUUUGCCUCAG 1436-1458 AD-52807.1 A-109049.1 UAUGUGUAAAAAUCUGUAAUA A-108677.2 UAUUACAGAUUUUUACACAUACU 1917-1939 AD-52808.1 A-109010.1 ACAAAGAUUUGGUGUUUUCUA A-108599.2 UAGAAAACACCAAAUCUUUGUUU 1176-1198 AD-52809.1 A-109018.1 UGUGGAGAAAACAACCUAAAU A-108615.2 AUUUAGGUUGUUUUCUCCACACU 1271-1293 AD-52810.1 A-109026.1 UGGAAGGUUAUACUCUAUAAA A-108631.2 UUUAUAGAGUAUAACCUUCCAUU 1363-1385 AD-52811.1 A-109034.1 AUGAACUGAGGCAAAUUUAAA A-108647.2 UUUAAAUUUGCCUCAGUUCAUUC 1429-1451 AD-52812.1 A-109042.1 AGGCAAAUUUAAAAGGCAAUA A-108663.2 UAUUGCCUUUUAAAUUUGCCUCA 1437-1459 AD-52813.1 A-109011.1 AAGAUUUGGUGUUUUCUACUU A-108601.2 AAGUAGAAAACACCAAAUCUUUG 1179-1201 AD-52814.1 A-109019.1 AAACAACCUAAAUGGUAAAUA A-108617.2 UAUUUACCAUUUAGGUUGUUUUC 1279-1301 AD-52815.1 A-109027.1 AUACUCUAUAAAAUCAACCAA A-108633.2 UUGGUUGAUUUUAUAGAGUAUAA 1372-1394 AD-52816.1 A-109035.1 UGAACUGAGGCAAAUUUAAAA A-108649.2 UUUUAAAUUUGCCUCAGUUCAUU 1430-1452 AD-52817.1 A-109043.1 GGCAAAUUUAAAAGGCAAUAA A-108665.2 UUAUUGCCUUUUAAAUUUGCCUC 1438-1460 AD-52818.1 A-109012.1 UUUUCUACUUGGGAUCACAAA A-108603.2 UUUGUGAUCCCAAGUAGAAAACA 1190-1212 AD-52819.1 A-109020.1 AACAACCUAAAUGGUAAAUAU A-108619.2 AUAUUUACCAUUUAGGUUGUUUU 1280-1302 AD-52820.1 A-109028.1 UACUCUAUAAAAUCAACCAAA A-108635.2 UUUGGUUGAUUUUAUAGAGUAUA 1373-1395 AD-52821.1 A-109036.1 GAACUGAGGCAAAUUUAAAAA A-108651.2 UUUUUAAAUUUGCCUCAGUUCAU 1431-1453_G21A AD-52822.1 A-109044.1 CAGAGUAUGUGUAAAAAUCUU A-108667.2 AAGAUUUUUACACAUACUCUGUG 1912-1934_G21U

TABLE 10 Modified Sense and antisense strand sequences of ANGPTL3 dsRNAs without GalNal conjugation These sequences are the same as the sequences listed in Table 8 except that they do not contain GalNal conjugation. Sense Sequence Antisense Oligo Sequence Sense (SEQ ID NOS 1366-1546, (SEQ ID NOS 1547-1727,  Duplex Oligo respectively, in Antisense respectively, in Name Name order of appearance) OligoName order of appearance) AD-52637.1 A-108817.1 UfcAfcAfaUfuAfAfGfcUfcCfuUfcUfuUf A-108307.2 aAfaGfaAfgGfaGfcuuAfaUfuGfuGfasAfsc AD-52638.1 A-108825.1 UfuAfuUfgUfuCfCfUfcUfaGfuUfaUfuUf A-108323.2 aAfaUfaAfcUfaGfaggAfaCfaAfuAfasAfsa AD-52639.1 A-108833.1 GfcUfaUfgUfuAfGfAfcGfaUfgUfaAfaAf A-108339.2 uUfuUfaCfaUfcGfucuAfaCfaUfaGfcsAfsa AD-52640.1 A-108841.1 GfgAfcAfuGfgUfCfUfuAfaAfgAfcUfuUf A-108355.2 aAfaGfuCfuUfuAfagaCfcAfuGfuCfcsCfsa AD-52641.1 A-108849.1 CfaAfaAfaCfuCfAfAfcAfuAfuUfuGfaUf A-108371.2 aUfcAfaAfuAfuGfuugAfgUfuUfuUfgsAfsa AD-52642.1 A-108857.1 AfcCfaGfuGfaAfAfUfcAfaAfgAfaGfaAf A-108387.2 uUfcUfuCfuUfuGfauuUfcAfcUfgGfusUfsu AD-52643.1 A-108818.1 CfaCfaAfuUfaAfGfCfuCfcUfuCfuUfuUf A-108309.2 aAfaAfgAfaGfgAfgcuUfaAfuUfgUfgsAfsa AD-52645.1 A-108834.1 CfuAfuGfuUfaGfAfCfgAfuGfuAfaAfaAf A-108341.2 uUfuUfuAfcAfuCfgucUfaAfcAfuAfgsCfsa AD-52647.1 A-108850.1 UfcAfaCfaUfaUfUfUfgAfuCfaGfuCfuUf A-108373.2 aAfgAfcUfgAfuCfaaaUfaUfgUfuGfasGfsu AD-52648.1 A-108858.1 AfaCfuGfaGfaAfGfAfaCfuAfcAfuAfuAf A-108389.2 uAfuAfuGfuAfgUfucuUfcUfcAfgUfusCfsc AD-52649.1 A-108819.1 AfcAfaUfuAfaGfCfUfcCfuUfcUfuUfuUf A-108311.2 aAfaAfaGfaAfgGfagcUfuAfaUfuGfusGfsa AD-52650.1 A-108827.1 CfuCfcAfgAfgCfCfAfaAfaUfcAfaGfaUf A-108327.2 aUfcUfuGfaUfuUfuggCfuCfuGfgAfgsAfsu AD-52651.1 A-108835.1 CfgAfuGfuAfaAfAfAfuUfuUfaGfcCfaAf A-108343.2 uUfgGfcUfaAfaAfuuuUfuAfcAfuCfgsUfsc AD-52652.1 A-108843.1 GfuCfuUfaAfaGfAfCfuUfuGfuCfcAfuAf A-108359.2 uAfuGfgAfcAfaAfgucUfuUfaAfgAfcsCfsa AD-52653.1 A-108851.1 CfaAfcAfuAfuUfUfGfaUfcAfgUfcUfuUf A-108375.2 aAfaGfaCfuGfaUfcaaAfuAfuGfuUfgsAfsg AD-52654.1 A-108859.1 AfcUfgAfgAfaGfAfAfcUfaCfaUfaUfaAf A-108391.2 uUfaUfaUfgUfaGfuucUfuCfuCfaGfusUfsc AD-52656.1 A-108828.1 CfcAfgAfgCfcAfAfAfaUfcAfaGfaUfuUf A-108329.2 aAfaUfcUfuGfaUfuuuGfgCfuCfuGfgsAfsg AD-52657.1 A-108836.1 GfaUfgUfaAfaAfAfUfuUfuAfgCfcAfaUf A-108345.2 aUfuGfgCfuAfaAfauuUfuUfaCfaUfcsGfsu AD-52658.1 A-108844.1 UfcUfuAfaAfgAfCfUfuUfgUfcCfaUfaAf A-108361.2 uUfaUfgGfaCfaAfaguCfuUfuAfaGfasCfsc AD-52659.1 A-108852.1 AfaCfaUfaUfuUfGfAfuCfaGfuCfuUfuUf A-108377.2 aAfaAfgAfcUfgAfucaAfaUfaUfgUfusGfsa AD-52660.1 A-108860.1 CfuGfaGfaAfgAfAfCfuAfcAfuAfuAfaAf A-108393.2 uUfuAfuAfuGfuAfguuCfuUfcUfcAfgsUfsu AD-52661.1 A-108821.1 AfaUfuAfaGfcUfCfCfuUfcUfuUfuUfaUf A-108315.2 aUfaAfaAfaGfaAfggaGfcUfuAfaUfusGfsu AD-52662.1 A-108829.1 AfaAfuCfaAfgAfUfUfuGfcUfaUfgUfuAf A-108331.2 uAfaCfaUfaGfcAfaauCfuUfgAfuUfusUfsg AD-52663.1 A-108837.1 UfuCfaGfuUfgGfGfAfcAfuGfgUfcUfuAf A-108347.2 uAfaGfaCfcAfuGfuccCfaAfcUfgAfasGfsg AD-52664.1 A-108845.1 GfgGfcCfaAfaUfUfAfaUfgAfcAfuAfuUf A-108363.2 aAfuAfuGfuCfaUfuaaUfuUfgGfcCfcsUfsu AD-52665.1 A-108853.1 AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUf A-108379.2 aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg AD-52666.1 A-108861.1 AfgAfaCfuAfcAfUfAfuAfaAfcUfaCfaAf A-108395.2 uUfgUfaGfuUfuAfuauGfuAfgUfuCfusUfsc AD-52667.1 A-108822.1 AfuUfaAfgCfuCfCfUfuCfuUfuUfuAfuUf A-108317.2 aAfuAfaAfaAfgAfaggAfgCfuUfaAfusUfsg AD-52668.1 A-108830.1 AfgAfuUfuGfcUfAfUfgUfuAfgAfcGfaUf A-108333.2 aUfcGfuCfuAfaCfauaGfcAfaAfuCfusUfsg AD-52669.1 A-108838.1 UfcAfgUfuGfgGfAfCfaUfgGfuCfuUfaAf A-108349.2 uUfaAfgAfcCfaUfgucCfcAfaCfuGfasAfsg AD-52670.1 A-108846.1 GfgCfcAfaAfuUfAfAfuGfaCfaUfaUfuUf A-108365.2 aAfaUfaUfgUfcAfuuaAfuUfuGfgCfcsCfsu AD-52671.1 A-108854.1 CfaUfaUfuUfgAfUfCfaGfuCfuUfuUfuAf A-108381.2 uAfaAfaAfgAfcUfgauCfaAfaUfaUfgsUfsu AD-52672.1 A-108862.1 UfaCfaUfaUfaAfAfCfuAfcAfaGfuCfaAf A-108397.2 uUfgAfcUfuGfuAfguuUfaUfaUfgUfasGfsu AD-52673.1 A-108823.1 UfuUfuAfuUfgUfUfCfcUfcUfaGfuUfaUf A-108319.2 aUfaAfcUfaGfaGfgaaCfaAfuAfaAfasAfsg AD-52674.1 A-108831.1 UfuGfcUfaUfgUfUfAfgAfcGfaUfgUfaAf A-108335.2 uUfaCfaUfcGfuCfuaaCfaUfaGfcAfasAfsu AD-52675.1 A-108839.1 CfaGfuUfgGfgAfCfAfuGfgUfcUfuAfaAf A-108351.2 uUfuAfaGfaCfcAfuguCfcCfaAfcUfgsAfsa AD-52676.1 A-108847.1 AfaAfuUfaAfuGfAfCfaUfaUfuUfcAfaAf A-108367.2 uUfuGfaAfaUfaUfgucAfuUfaAfuUfusGfsg AD-52677.1 A-108855.1 GfaUfcAfgUfcUfUfUfuUfaUfgAfuCfuAf A-108383.2 uAfgAfuCfaUfaAfaaaGfaCfuGfaUfcsAfsa AD-52678.1 A-108863.1 AfcAfuAfuAfaAfCfUfaCfaAfgUfcAfaAf A-108399.2 uUfuGfaCfuUfgUfaguUfuAfuAfuGfusAfsg AD-52679.1 A-108824.1 UfuUfaUfuGfuUfCfCfuCfuAfgUfuAfuUf A-108321.2 aAfuAfaCfuAfgAfggaAfcAfaUfaAfasAfsa AD-52680.1 A-108832.1 UfgCfuAfuGfuUfAfGfaCfgAfuGfuAfaAf A-108337.2 uUfuAfcAfuCfgUfcuaAfcAfuAfgCfasAfsa AD-52681.1 A-108840.1 GfgGfaCfaUfgGfUfCfuUfaAfaGfaCfuUf A-108353.2 aAfgUfcUfuUfaAfgacCfaUfgUfcCfcsAfsa AD-52682.1 A-108848.1 UfgAfcAfuAfuUfUfCfaAfaAfaCfuCfaAf A-108369.2 uUfgAfgUfuUfuUfgaaAfuAfuGfuCfasUfsu AD-52683.1 A-108856.1 AfuCfaGfuCfuUfUfUfuAfuGfaUfcUfaUf A-108385.2 aUfaGfaUfcAfuAfaaaAfgAfcUfgAfusCfsa AD-52684.1 A-108864.1 CfaUfaUfaAfaCfUfAfcAfaGfuCfaAfaAf A-108401.2 uUfuUfgAfcUfuGfuagUfuUfaUfaUfgsUfsa AD-52685.1 A-108872.1 CfuUfgAfaCfuCfAfAfcUfcAfaAfaCfuUf A-108417.2 aAfgUfuUfuGfaGfuugAfgUfuCfaAfgsUfsg AD-52686.1 A-108880.1 CfuAfcUfuCfaAfCfAfaAfaAfgUfgAfaAf A-108433.2 uUfuCfaCfuUfuUfuguUfgAfaGfuAfgsAfsa AD-52687.1 A-108888.1 AfaGfaGfcAfaCfUfAfaCfuAfaCfuUfaAf A-108449.2 uUfaAfgUfuAfgUfuagUfuGfcUfcUfusCfsu AD-52688.1 A-108896.1 AfaAfcAfaGfaUfAfAfuAfgCfaUfcAfaAf A-108465.2 uUfuGfaUfgCfuAfuuaUfcUfuGfuUfusUfsu AD-52689.1 A-108904.1 GfcAfuAfgUfcAfAfAfuAfaAfaGfaAfaUf A-108481.2 aUfuUfcUfuUfuAfuuuGfaCfuAfuGfcsUfsg AD-52690.1 A-108865.1 AfuAfuAfaAfcUfAfCfaAfgUfcAfaAfaAf A-108403.2 uUfuUfuGfaCfuUfguaGfuUfuAfuAfusGfsu AD-52691.1 A-108873.1 GfaAfcUfcAfaCfUfCfaAfaAfcUfuGfaAf A-108419.2 uUfcAfaGfuUfuUfgagUfuGfaGfuUfcsAfsa AD-52692.1 A-108881.1 UfaCfuUfcAfaCfAfAfaAfaGfuGfaAfaUf A-108435.2 aUfuUfcAfcUfuUfuugUfuGfaAfgUfasGfsa AD-52693.1 A-108889.1 AfgAfgCfaAfcUfAfAfcUfaAfcUfuAfaUf A-108451.2 aUfuAfaGfuUfaGfuuaGfuUfgCfuCfusUfsc AD-52694.1 A-108897.1 GfaUfaAfuAfgCfAfUfcAfaAfgAfcCfuUf A-108467.2 aAfgGfuCfuUfuGfaugCfuAfuUfaUfcsUfsu AD-52695.1 A-108905.1 CfaUfaGfuCfaAfAfUfaAfaAfgAfaAfuAf A-108483.2 uAfuUfuCfuUfuUfauuUfgAfcUfaUfgsCfsu AD-52696.1 A-108866.1 UfaUfaAfaCfuAfCfAfaGfuCfaAfaAfaUf A-108405.2 aUfuUfuUfgAfcUfuguAfgUfuUfaUfasUfsg AD-52697.1 A-108874.1 AfaCfuCfaAfcUfCfAfaAfaCfuUfgAfaAf A-108421.2 uUfuCfaAfgUfuUfugaGfuUfgAfgUfusCfsa AD-52698.1 A-108882.1 AfcUfuCfaAfcAfAfAfaAfgUfgAfaAfuAf A-108437.2 uAfuUfuCfaCfuUfuuuGfuUfgAfaGfusAfsg AD-52699.1 A-108890.1 GfaGfcAfaCfuAfAfCfuAfaCfuUfaAfuUf A-108453.2 aAfuUfaAfgUfuAfguuAfgUfuGfcUfcsUfsu AD-52700.1 A-108898.1 AfaCfcAfaCfaGfCfAfuAfgUfcAfaAfuAf A-108469.2 uAfuUfuGfaCfuAfugcUfgUfuGfgUfusUfsa AD-52701.1 A-108906.1 AfgUfcAfaAfuAfAfAfaGfaAfaUfaGfaAf A-108485.2 uUfcUfaUfuUfcUfuuuAfuUfuGfaCfusAfsu AD-52702.1 A-108867.1 AfgUfcAfaAfaAfUfGfaAfgAfgGfuAfaAf A-108407.2 uUfuAfcCfuCfuUfcauUfuUfuGfaCfusUfsg AD-52703.1 A-108875.1 CfuUfgAfaAfgCfCfUfcCfuAfgAfaGfaAf A-108423.2 uUfcUfuCfuAfgGfaggCfuUfuCfaAfgsUfsu AD-52704.1 A-108883.1 CfuUfcAfaCfaAfAfAfaGfuGfaAfaUfaUf A-108439.2 aUfaUfuUfcAfcUfuuuUfgUfuGfaAfgsUfsa AD-52705.1 A-108891.1 CfaAfcUfaAfcUfAfAfcUfuAfaUfuCfaAf A-108455.2 uUfgAfaUfuAfaGfuuaGfuUfaGfuUfgsCfsu AD-52706.1 A-108899.1 AfcCfaAfcAfgCfAfUfaGfuCfaAfaUfaAf A-108471.2 uUfaUfuUfgAfcUfaugCfuGfuUfgGfusUfsu AD-52707.1 A-108907.1 GfaAfcCfcAfcAfGfAfaAfuUfuCfuCfuAf A-108487.2 uAfgAfgAfaAfuUfucuGfuGfgGfuUfcsUfsu AD-52708.1 A-108868.1 GfaAfuAfuGfuCfAfCfuUfgAfaCfuCfaAf A-108409.2 uUfgAfgUfuCfaAfgugAfcAfuAfuUfcsUfsu AD-52709.1 A-108876.1 UfgAfaAfgCfcUfCfCfuAfgAfaGfaAfaAf A-108425.2 uUfuUfcUfuCfuAfggaGfgCfuUfuCfasAfsg AD-52710.1 A-108884.1 UfuCfaAfcAfaAfAfAfgUfgAfaAfuAfuUf A-108441.2 aAfuAfuUfuCfaCfuuuUfuGfuUfgAfasGfsu AD-52711.1 A-108892.1 AfaCfuAfaCfuAfAfCfuUfaAfuUfcAfaAf A-108457.2 uUfuGfaAfuUfaAfguuAfgUfuAfgUfusGfsc AD-52712.1 A-108900.1 CfcAfaCfaGfcAfUfAfgUfcAfaAfuAfaAf A-108473.2 uUfuAfuUfuGfaCfuauGfcUfgUfuGfgsUfsu AD-52713.1 A-108908.1 AfaCfcCfaCfaGfAfAfaUfuUfcUfcUfaUf A-108489.2 aUfaGfaGfaAfaUfuucUfgUfgGfgUfusCfsu AD-52714.1 A-108869.1 UfgUfcAfcUfuGfAfAfcUfcAfaCfuCfaAf A-108411.2 uUfgAfgUfuGfaGfuucAfaGfuGfaCfasUfsa AD-52715.1 A-108877.1 GfaAfaGfcCfuCfCfUfaGfaAfgAfaAfaAf A-108427.2 uUfuUfuCfuUfcUfaggAfgGfcUfuUfcsAfsa AD-52716.1 A-108885.1 AfaUfaUfuUfaGfAfAfgAfgCfaAfcUfaAf A-108443.2 uUfaGfuUfgCfuCfuucUfaAfaUfaUfusUfsc AD-52717.1 A-108893.1 AfcUfaAfcUfaAfCfUfuAfaUfuCfaAfaAf A-108459.2 uUfuUfgAfaUfuAfaguUfaGfuUfaGfusUfsg AD-52718.1 A-108901.1 CfaAfcAfgCfaUfAfGfuCfaAfaUfaAfaAf A-108475.2 uUfuUfaUfuUfgAfcuaUfgCfuGfuUfgsGfsu AD-52719.1 A-108909.1 CfcAfcAfgAfaAfUfUfuCfuCfuAfuCfuUf A-108491.2 aAfgAfuAfgAfgAfaauUfuCfuGfuGfgsGfsu AD-52720.1 A-108870.1 GfuCfaCfuUfgAfAfCfuCfaAfcUfcAfaAf A-108413.2 uUfuGfaGfuUfgAfguuCfaAfgUfgAfcsAfsu AD-52721.1 A-108878.1 CfuCfcUfaGfaAfGfAfaAfaAfaUfuCfuAf A-108429.2 uAfgAfaUfuUfuUfucuUfcUfaGfgAfgsGfsc AD-52722.1 A-108886.1 AfuUfuAfgAfaGfAfGfcAfaCfuAfaCfuAf A-108445.2 uAfgUfuAfgUfuGfcucUfuCfuAfaAfusAfsu AD-52723.1 A-108894.1 CfuAfaCfuAfaCfUfUfaAfuUfcAfaAfaUf A-108461.2 aUfuUfuGfaAfuUfaagUfuAfgUfuAfgsUfsu AD-52724.1 A-108902.1 CfaGfcAfuAfgUfCfAfaAfuAfaAfaGfaAf A-108477.2 uUfcUfuUfuAfuUfugaCfuAfuGfcUfgsUfsu AD-52725.1 A-108910.1 GfaAfaUfaAfgAfAfAfuGfuAfaAfaCfaUf A-108493.2 aUfgUfuUfuAfcAfuuuCfuUfaUfuUfcsAfsu AD-52726.1 A-108871.1 UfcAfcUfuGfaAfCfUfcAfaCfuCfaAfaAf A-108415.2 uUfuUfgAfgUfuGfaguUfcAfaGfuGfasCfsa AD-52727.1 A-108879.1 UfcUfaCfuUfcAfAfCfaAfaAfaGfuGfaAf A-108431.2 uUfcAfcUfuUfuUfguuGfaAfgUfaGfasAfsu AD-52728.1 A-108887.1 UfuUfaGfaAfgAfGfCfaAfcUfaAfcUfaAf A-108447.2 uUfaGfuUfaGfuUfgcuCfuUfcUfaAfasUfsa AD-52729.1 A-108895.1 AfaAfaCfaAfgAfUfAfaUfaGfcAfuCfaAf A-108463.2 uUfgAfuGfcUfaUfuauCfuUfgUfuUfusUfsc AD-52730.1 A-108903.1 AfgCfaUfaGfuCfAfAfaUfaAfaAfgAfaAf A-108479.2 uUfuCfuUfuUfaUfuugAfcUfaUfgCfusGfsu AD-52731.1 A-108958.1 AfgAfcCfcAfgCfAfAfcUfcUfcAfaGfuUf A-108495.2 aAfcUfuGfaGfaGfuugCfuGfgGfuCfusGfsa AD-52732.1 A-108966.1 AfgUfcCfaUfgGfAfCfaUfuAfaUfuCfaAf A-108511.2 uUfgAfaUfuAfaUfgucCfaUfgGfaCfusAfsc AD-52733.1 A-108974.1 GfaUfgGfaUfcAfCfAfaAfaCfuUfcAfaUf A-108527.2 aUfuGfaAfgUfuUfuguGfaUfcCfaUfcsUfsa AD-52734.1 A-108982.1 CfuAfgAfgAfaGfAfUfaUfaCfuCfcAfuAf A-108543.2 uAfuGfgAfgUfaUfaucUfuCfuCfuAfgsGfsc AD-52735.1 A-108990.1 AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf A-108559.2 aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc AD-52736.1 A-108998.1 CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUf A-108575.2 aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu AD-52737.1 A-108959.1 GfaCfcCfaGfcAfAfCfuCfuCfaAfgUfuUf A-108497.2 aAfaCfuUfgAfgAfguuGfcUfgGfgUfcsUfsg AD-52739.1 A-108975.1 GfgAfuCfaCfaAfAfAfcUfuCfaAfuGfaAf A-108529.2 uUfcAfuUfgAfaGfuuuUfgUfgAfuCfcsAfsu AD-52740.1 A-108983.1 GfaAfgAfuAfuAfCfUfcCfaUfaGfuGfaAf A-108545.2 uUfcAfcUfaUfgGfaguAfuAfuCfuUfcsUfsc AD-52741.1 A-108991.1 GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf A-108561.2 uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu AD-52742.1 A-108999.1 GfgGfaAfaUfcAfCfGfaAfaCfcAfaCfuAf A-108577.2 uAfgUfuGfgUfuUfcguGfaUfuUfcCfcsAfsa AD-52743.1 A-108960.1 AfcCfcAfgCfaAfCfUfcUfcAfaGfuUfuUf A-108499.2 aAfaAfcUfuGfaGfaguUfgCfuGfgGfusCfsu AD-52744.1 A-108968.1 GfgAfcAfuUfaAfUfUfcAfaCfaUfcGfaAf A-108515.2 uUfcGfaUfgUfuGfaauUfaAfuGfuCfcsAfsu AD-52745.1 A-108976.1 GfaUfcAfcAfaAfAfCfuUfcAfaUfgAfaAf A-108531.2 uUfuCfaUfuGfaAfguuUfuGfuGfaUfcsCfsa AD-52746.1 A-108984.1 AfcUfcCfaUfaGfUfGfaAfgCfaAfuCfuAf A-108547.2 uAfgAfuUfgCfuUfcacUfaUfgGfaGfusAfsu AD-52747.1 A-108992.1 AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf A-108563.2 aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu AD-52748.1 A-109000.1 GfgAfaAfuCfaCfGfAfaAfcCfaAfcUfaUf A-108579.2 aUfaGfuUfgGfuUfucgUfgAfuUfuCfcsCfsa AD-52749.1 A-108961.1 CfcCfaGfcAfaCfUfCfuCfaAfgUfuUfuUf A-108501.2 aAfaAfaCfuUfgAfgagUfuGfcUfgGfgsUfsc AD-52750.1 A-108969.1 GfaCfaUfuAfaUfUfCfaAfcAfuCfgAfaUf A-108517.2 aUfuCfgAfuGfuUfgaaUfuAfaUfgUfcsCfsa AD-52751.1 A-108977.1 AfaCfgUfgGfgAfGfAfaCfuAfcAfaAfuAf A-108533.2 uAfuUfuGfuAfgUfucuCfcCfaCfgUfusUfsc AD-52752.1 A-108985.1 CfuCfcAfuAfgUfGfAfaGfcAfaUfcUfaAf A-108549.2 uUfaGfaUfuGfcUfucaCfuAfuGfgAfgsUfsa AD-52753.1 A-108993.1 CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAf A-108565.2 uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc AD-52754.1 A-109001.1 GfaAfaUfcAfcGfAfAfaCfcAfaCfuAfuAf A-108581.2 uAfuAfgUfuGfgUfuucGfuGfaUfuUfcsCfsc AD-52755.1 A-108962.1 CfuCfuCfaAfgUfUfUfuUfcAfuGfuCfuAf A-108503.2 uAfgAfcAfuGfaAfaaaCfuUfgAfgAfgsUfsu AD-52756.1 A-108970.1 AfcAfuUfaAfuUfCfAfaCfaUfcGfaAfuAf A-108519.2 uAfuUfcGfaUfgUfugaAfuUfaAfuGfusCfsc AD-52757.1 A-108978.1 GfgGfaGfaAfcUfAfCfaAfaUfaUfgGfuUf A-108535.2 aAfcCfaUfaUfuUfguaGfuUfcUfcCfcsAfsc AD-52758.1 A-108986.1 UfcCfaUfaGfuGfAfAfgCfaAfuCfuAfaUf A-108551.2 aUfuAfgAfuUfgCfuucAfcUfaUfgGfasGfsu AD-52759.1 A-108994.1 AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUf A-108567.2 aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu AD-52760.1 A-109002.1 UfgGfcAfaUfgUfCfCfcCfaAfuGfcAfaUf A-108583.2 aUfuGfcAfuUfgGfggaCfaUfuGfcCfasGfsu AD-52761.1 A-108963.1 UfcAfgGfuAfgUfCfCfaUfgGfaCfaUfuAf A-108505.2 uAfaUfgUfcCfaUfggaCfuAfcCfuGfasUfsa AD-52762.1 A-108971.1 UfuAfaUfuCfaAfCfAfuCfgAfaUfaGfaUf A-108521.2 aUfcUfaUfuCfgAfuguUfgAfaUfuAfasUfsg AD-52763.1 A-108979.1 GfgAfgAfaCfuAfCfAfaAfuAfuGfgUfuUf A-108537.2 aAfaCfcAfuAfuUfuguAfgUfuCfuCfcsCfsa AD-52764.1 A-108987.1 CfcAfuAfgUfgAfAfGfcAfaUfcUfaAfuUf A-108553.2 aAfuUfaGfaUfuGfcuuCfaCfuAfuGfgsAfsg AD-52765.1 A-108995.1 AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUf A-108569.2 aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg AD-52766.1 A-109003.1 AfaUfgCfaAfuCfCfCfgGfaAfaAfcAfaAf A-108585.2 uUfuGfuUfuUfcCfgggAfuUfgCfaUfusGfsg AD-52767.1 A-108964.1 CfaGfgUfaGfuCfCfAfuGfgAfcAfuUfaAf A-108507.2 uUfaAfuGfuCfcAfuggAfcUfaCfcUfgsAfsu AD-52768.1 A-108972.1 UfuCfaAfcAfuCfGfAfaUfaGfaUfgGfaUf A-108523.2 aUfcCfaUfcUfaUfucgAfuGfuUfgAfasUfsu AD-52769.1 A-108980.1 GfuUfgGfgCfcUfAfGfaGfaAfgAfuAfuAf A-108539.2 uAfuAfuCfuUfcUfcuaGfgCfcCfaAfcsCfsa AD-52770.1 A-108988.1 CfaUfaGfuGfaAfGfCfaAfuCfuAfaUfuAf A-108555.2 uAfaUfuAfgAfuUfgcuUfcAfcUfaUfgsGfsa AD-52771.1 A-108996.1 AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf A-108571.2 aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg AD-52772.1 A-109004.1 GfcAfaUfcCfcGfGfAfaAfaCfaAfaGfaUf A-108587.2 aUfcUfuUfgUfuUfuccGfgGfaUfuGfcsAfsu AD-52773.1 A-108965.1 GfgUfaGfuCfcAfUfGfgAfcAfuUfaAfuUf A-108509.2 aAfuUfaAfuGfuCfcauGfgAfcUfaCfcsUfsg AD-52774.1 A-108973.1 AfuCfgAfaUfaGfAfUfgGfaUfcAfcAfaAf A-108525.2 uUfuGfuGfaUfcCfaucUfaUfuCfgAfusGfsu AD-52775.1 A-108981.1 CfcUfaGfaGfaAfGfAfuAfuAfcUfcCfaUf A-108541.2 aUfgGfaGfuAfuAfucuUfcUfcUfaGfgsCfsc AD-52776.1 A-108989.1 GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAf A-108557.2 uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc AD-52777.1 A-108997.1 AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUf A-108573.2 aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu AD-52778.1 A-109005.1 CfaAfuCfcCfgGfAfAfaAfcAfaAfgAfuUf A-108589.2 aAfuCfuUfuGfuUfuucCfgGfgAfuUfgsCfsa AD-52779.1 A-109013.1 CfuAfcUfuGfgGfAfUfcAfcAfaAfgCfaAf A-108605.2 uUfgCfuUfuGfuGfaucCfcAfaGfuAfgsAfsa AD-52780.1 A-109021.1 AfcAfaCfcUfaAfAfUfgGfuAfaAfuAfuAf A-108621.2 uAfuAfuUfuAfcCfauuUfaGfgUfuGfusUfsu AD-52781.1 A-109029.1 AfuCfcAfuCfcAfAfCfaGfaUfuCfaGfaAf A-108637.2 uUfcUfgAfaUfcUfguuGfgAfuGfgAfusCfsa AD-52782.1 A-109037.1 AfaCfuGfaGfgCfAfAfaUfuUfaAfaAfgAf A-108653.2 uCfuUfuUfaAfaUfuugCfcUfcAfgUfusCfsa AD-52783.1 A-109045.1 AfgAfgUfaUfgUfGfUfaAfaAfaUfcUfgUf A-108669.2 aCfaGfaUfuUfuUfacaCfaUfaCfuCfusGfsu AD-52784.1 A-109006.1 AfaUfcCfcGfgAfAfAfaCfaAfaGfaUfuUf A-108591.2 aAfaUfcUfuUfgUfuuuCfcGfgGfaUfusGfsc AD-52785.1 A-109014.1 UfaCfuUfgGfgAfUfCfaCfaAfaGfcAfaAf A-108607.2 uUfuGfcUfuUfgUfgauCfcCfaAfgUfasGfsa AD-52786.1 A-109022.1 CfaAfcCfuAfaAfUfGfgUfaAfaUfaUfaAf A-108623.2 uUfaUfaUfuUfaCfcauUfuAfgGfuUfgsUfsu AD-52787.1 A-109030.1 UfuGfaAfuGfaAfCfUfgAfgGfcAfaAfuUf A-108639.2 aAfuUfuGfcCfuCfaguUfcAfuUfcAfasAfsg AD-52788.1 A-109038.1 AfcUfgAfgGfcAfAfAfuUfuAfaAfaGfgAf A-108655.2 uCfcUfuUfuAfaAfuuuGfcCfuCfaGfusUfsc AD-52789.1 A-109046.1 GfaGfuAfuGfuGfUfAfaAfaAfuCfuGfuAf A-108671.2 uAfcAfgAfuUfuUfuacAfcAfuAfcUfcsUfsg AD-52791.1 A-109015.1 AfcUfuGfgGfaUfCfAfcAfaAfgCfaAfaAf A-108609.2 uUfuUfgCfuUfuGfugaUfcCfcAfaGfusAfsg AD-52792.1 A-109023.1 AfuGfgUfaAfaUfAfUfaAfcAfaAfcCfaAf A-108625.2 uUfgGfuUfuGfuUfauaUfuUfaCfcAfusUfsu AD-52793.1 A-109031.1 UfgAfaUfgAfaCfUfGfaGfgCfaAfaUfuUf A-108641.2 aAfaUfuUfgCfcUfcagUfuCfaUfuCfasAfsa AD-52794.1 A-109039.1 CfuGfaGfgCfaAfAfUfuUfaAfaAfgGfcAf A-108657.2 uGfcCfuUfuUfaAfauuUfgCfcUfcAfgsUfsu AD-52795.1 A-109047.1 AfgUfaUfgUfgUfAfAfaAfaUfcUfgUfaAf A-108673.2 uUfaCfaGfaUfuUfuuaCfaCfaUfaCfusCfsu AD-52796.1 A-109008.1 GfaAfaAfcAfaAfGfAfuUfuGfgUfgUfuUf A-108595.2 aAfaCfaCfcAfaAfucuUfuGfuUfuUfcsCfsg AD-52797.1 A-109016.1 AfgUfgUfgGfaGfAfAfaAfcAfaCfcUfaAf A-108611.2 uUfaGfgUfuGfuUfuucUfcCfaCfaCfusCfsa AD-52798.1 A-109024.1 GfuCfuCfaAfaAfUfGfgAfaGfgUfuAfuAf A-108627.2 uAfuAfaCfcUfuCfcauUfuUfgAfgAfcsUfsu AD-52799.1 A-109032.1 GfaAfuGfaAfcUfGfAfgGfcAfaAfuUfuAf A-108643.2 uAfaAfuUfuGfcCfucaGfuUfcAfuUfcsAfsa AD-52800.1 A-109040.1 UfgAfgGfcAfaAfUfUfuAfaAfaGfgCfaAf A-108659.2 uUfgCfcUfuUfuAfaauUfuGfcCfuCfasGfsu AD-52801.1 A-109048.1 GfuAfuGfuGfuAfAfAfaAfuCfuGfuAfaUf A-108675.2 aUfuAfcAfgAfuUfuuuAfcAfcAfuAfcsUfsc AD-52802.1 A-109009.1 AfaAfaCfaAfaGfAfUfuUfgGfuGfuUfuUf A-108597.2 aAfaAfcAfcCfaAfaucUfuUfgUfuUfusCfsc AD-52803.1 A-109017.1 GfuGfuGfgAfgAfAfAfaCfaAfcCfuAfaAf A-108613.2 uUfuAfgGfuUfgUfuuuCfuCfcAfcAfcsUfsc AD-52804.1 A-109025.1 AfuGfgAfaGfgUfUfAfuAfcUfcUfaUfaAf A-108629.2 uUfaUfaGfaGfuAfuaaCfcUfuCfcAfusUfsu AD-52805.1 A-109033.1 AfaUfgAfaCfuGfAfGfgCfaAfaUfuUfaAf A-108645.2 uUfaAfaUfuUfgCfcucAfgUfuCfaUfusCfsa AD-52806.1 A-109041.1 GfaGfgCfaAfaUfUfUfaAfaAfgGfcAfaUf A-108661.2 aUfuGfcCfuUfuUfaaaUfuUfgCfcUfcsAfsg AD-52807.1 A-109049.1 UfaUfgUfgUfaAfAfAfaUfcUfgUfaAfuAf A-108677.2 uAfuUfaCfaGfaUfuuuUfaCfaCfaUfasCfsu AD-52808.1 A-109010.1 AfcAfaAfgAfuUfUfGfgUfgUfuUfuCfuAf A-108599.2 uAfgAfaAfaCfaCfcaaAfuCfuUfuGfusUfsu AD-52809.1 A-109018.1 UfgUfgGfaGfaAfAfAfcAfaCfcUfaAfaUf A-108615.2 aUfuUfaGfgUfuGfuuuUfcUfcCfaCfasCfsu AD-52810.1 A-109026.1 UfgGfaAfgGfuUfAfUfaCfuCfuAfuAfaAf A-108631.2 uUfuAfuAfgAfgUfauaAfcCfuUfcCfasUfsu AD-52811.1 A-109034.1 AfuGfaAfcUfgAfGfGfcAfaAfuUfuAfaAf A-108647.2 uUfuAfaAfuUfuGfccuCfaGfuUfcAfusUfsc AD-52812.1 A-109042.1 AfgGfcAfaAfuUfUfAfaAfaGfgCfaAfuAf A-108663.2 uAfuUfgCfcUfuUfuaaAfuUfuGfcCfusCfsa AD-52813.1 A-109011.1 AfaGfaUfuUfgGfUfGfuUfuUfcUfaCfuUf A-108601.2 aAfgUfaGfaAfaAfcacCfaAfaUfcUfusUfsg AD-52814.1 A-109019.1 AfaAfcAfaCfcUfAfAfaUfgGfuAfaAfuAf A-108617.2 uAfuUfuAfcCfaUfuuaGfgUfuGfuUfusUfsc AD-52815.1 A-109027.1 AfuAfcUfcUfaUfAfAfaAfuCfaAfcCfaAf A-108633.2 uUfgGfuUfgAfuUfuuaUfaGfaGfuAfusAfsa AD-52816.1 A-109035.1 UfgAfaCfuGfaGfGfCfaAfaUfuUfaAfaAf A-108649.2 uUfuUfaAfaUfuUfgccUfcAfgUfuCfasUfsu AD-52817.1 A-109043.1 GfgCfaAfaUfuUfAfAfaAfgGfcAfaUfaAf A-108665.2 uUfaUfuGfcCfuUfuuaAfaUfuUfgCfcsUfsc AD-52818.1 A-109012.1 UfuUfuCfuAfcUfUfGfgGfaUfcAfcAfaAf A-108603.2 uUfuGfuGfaUfcCfcaaGfuAfgAfaAfasCfsa AD-52819.1 A-109020.1 AfaCfaAfcCfuAfAfAfuGfgUfaAfaUfaUf A-108619.2 aUfaUfuUfaCfcAfuuuAfgGfuUfgUfusUfsu AD-52820.1 A-109028.1 UfaCfuCfuAfuAfAfAfaUfcAfaCfcAfaAf A-108635.2 uUfuGfgUfuGfaUfuuuAfuAfgAfgUfasUfsa AD-52821.1 A-109036.1 GfaAfcUfgAfgGfCfAfaAfuUfuAfaAfaAf A-108651.2 uUfuUfuAfaAfuUfugcCfuCfaGfuUfcsAfsu AD-52822.1 A-109044.1 CfaGfaGfuAfuGfUfGfuAfaAfaAfuCfuUf A-108667.2 aAfgAfuUfuUfuAfcacAfuAfcUfcUfgsUfsg

TABLE 11 Results of single dose screen using ANGPTL3 GalNac-conjugated dsRNA Modified siRNAs were tested by transfection in Hep3b cells and by free- uptake in primary cynomolgus monkey (PCH) cells at the above-stated doses. 500 nM 100 nM 10 nM PCH PCH PCH STDEV STDEV STDEV STDEV STDEV 10 nM 0.1 nM Celsis Celsis Celsis 10 nM 0.1 nM 500 nM 100 nM 10 nM DUPLEX ID (RNAimax) (RNAimax) (FU) (FU) (FU) (RNAimax) (RNAimax) (FU) (FU) (FU) AD1955/naïve FU 0.93 0.93 1.01 0.91 1.17 0.02 0.08 0.09 0.00 0.07 AD1955/naïve FU 1.02 1.09 1.07 1.07 0.92 0.06 0.04 0.02 0.00 0.03 AD1955/naïve FU 1.06 0.99 0.93 1.02 0.93 0.03 0.00 0.09 0.01 0.02 AD1955/naïve FU 1.05 0.90 1.05 1.03 1.03 0.04 0.02 0.01 0.05 0.01 AD1955/naïve FU 1.06 1.08 0.90 0.97 1.03 0.02 0.01 0.02 0.04 0.09 AD1955/naïve FU 0.90 1.03 1.05 1.00 0.94 0.04 0.03 0.01 0.04 0.05 AD-45165 (TTR) 0.91 0.98 1.06 0.98 0.96 0.05 0.01 0.05 0.00 0.00 AD-52953.1 0.06 0.34 0.15 0.17 0.46 0.00 0.01 0.00 0.01 0.01 AD-52954.1 0.09 0.39 0.17 0.20 0.55 0.00 0.01 0.00 0.01 0.00 AD-52955.1 0.11 0.59 0.38 0.41 0.75 0.01 0.04 0.02 0.01 0.12 AD-52956.1 0.31 0.94 0.79 0.94 1.17 0.01 0.00 0.02 0.06 0.02 AD-52957.1 0.13 0.61 0.35 0.38 0.73 0.01 0.00 0.01 0.00 0.04 AD-52958.1 0.19 0.74 0.66 0.71 0.97 0.01 0.01 0.02 0.07 0.06 AD-52960.1 0.14 0.59 0.31 0.32 0.55 0.01 0.01 0.00 0.02 0.02 AD-52961.1 0.05 0.66 0.27 0.24 0.49 0.00 0.00 0.00 0.02 0.02 AD-52962.1 0.83 0.89 1.03 1.02 1.26 0.02 0.05 0.07 0.07 0.07 AD-52963.1 0.07 0.72 0.46 0.56 0.91 0.00 0.00 0.00 0.00 0.06 AD-52964.1 0.13 0.73 0.41 0.47 0.68 0.01 0.03 0.02 0.03 0.01 AD-52965.1 0.07 0.44 0.16 0.18 0.43 0.00 0.01 0.00 0.01 0.01 AD-52966.1 0.12 0.76 0.67 0.72 0.96 0.00 0.02 0.05 0.01 0.01 AD-52967.1 0.10 0.75 0.44 0.58 0.89 0.01 0.04 0.02 0.03 0.04 AD-52968.1 1.01 0.96 0.87 0.91 1.15 0.00 0.01 0.09 0.03 0.02 AD-52969.1 0.04 0.46 0.22 0.29 0.59 0.00 0.00 0.01 0.02 0.04 AD-52970.1 0.06 0.45 0.27 0.30 0.51 0.00 0.00 0.01 0.02 0.00 AD-52971.1 0.08 0.55 0.20 0.22 0.45 0.00 0.00 0.01 0.02 0.05 AD-52972.1 0.10 0.73 0.41 0.49 0.81 0.00 0.01 0.01 0.02 0.01 AD-52973.1 0.11 0.73 0.36 0.46 0.75 0.01 0.01 0.03 0.02 0.02 AD-52974.1 1.00 0.95 1.00 1.09 1.27 0.01 0.01 0.08 0.05 0.06 AD-52975.1 0.07 0.54 0.25 0.34 0.66 0.00 0.01 0.01 0.01 0.03 AD-52976.1 0.17 0.59 0.35 0.41 0.65 0.00 0.02 0.04 0.01 0.01 AD-52977.1 0.07 0.45 0.16 0.25 0.50 0.01 0.02 0.00 0.02 0.03 AD-52978.1 0.10 0.72 0.39 0.53 0.77 0.00 0.02 0.00 0.08 0.03 AD-52979.1 0.54 0.92 0.99 1.12 1.28 0.01 0.02 0.02 0.04 0.05 AD-52980.1 0.29 0.85 0.67 0.85 1.03 0.01 0.01 0.05 0.05 0.04 AD-52981.1 0.07 0.44 0.20 0.26 0.59 0.01 0.02 0.00 0.00 0.03 AD-52982.1 0.28 0.87 0.67 0.99 1.14 0.01 0.01 0.04 0.00 0.01 AD-52983.1 0.06 0.40 0.14 0.40 0.46 0.00 0.00 0.01 0.05 0.02 AD-52984.1 0.29 0.87 0.66 0.74 1.09 0.01 0.02 0.01 0.00 0.00 AD-52985.1 0.72 0.87 0.89 1.18 1.22 0.03 0.00 0.05 0.03 0.16 AD-52986.1 0.08 0.47 0.24 0.30 0.48 0.00 0.02 0.02 0.00 0.06 AD-52987.1 0.16 0.83 0.42 0.73 1.09 0.00 0.00 0.01 0.02 0.02 AD-52988.1 0.11 0.73 0.42 0.60 0.96 0.01 0.04 0.00 0.00 0.10 AD-52989.1 0.05 0.48 0.15 0.42 0.46 0.00 0.02 0.00 0.02 0.00 AD-52990.1 0.14 0.86 0.33 0.45 0.77 0.00 0.01 0.00 0.02 0.05 AD-52991.1 0.16 0.86 0.58 0.69 1.05 0.00 0.00 0.02 0.00 0.02 AD-52992.1 0.08 0.65 0.42 0.56 0.90 0.00 0.01 0.02 0.01 0.00 AD-52993.1 0.13 0.87 0.53 0.76 1.08 0.02 0.03 0.04 0.04 0.00 AD-52994.1 0.10 0.52 0.28 0.33 0.53 0.01 0.00 0.02 0.00 0.01 AD-52995.1 0.06 0.56 0.19 0.41 0.60 0.00 0.01 0.04 0.02 0.05 AD-52996.1 0.09 0.68 0.26 0.47 0.68 0.00 0.03 0.01 0.04 0.01 AD-52997.1 0.59 1.03 0.87 0.51 1.25 0.05 0.01 0.00 0.01 0.01 AD-52998.1 0.09 0.79 0.44 0.55 0.85 0.00 0.00 0.04 0.03 0.10 AD-52999.1 0.08 0.57 0.17 0.36 0.84 0.01 0.00 0.01 0.02 0.00 AD-53000.1 0.38 0.94 0.58 0.67 0.85 0.01 0.02 0.03 0.03 0.02 AD-53001.1 0.05 0.48 0.21 0.18 0.40 0.00 0.00 0.01 0.00 0.05 AD-53002.1 0.07 0.65 0.43 0.48 0.80 0.00 0.05 0.04 0.01 0.02 AD-53003.1 0.05 0.46 0.31 0.34 0.56 0.01 0.01 0.00 0.02 0.05 AD-53004.1 0.05 0.36 0.29 0.66 0.57 0.00 0.01 0.03 0.35 0.02 AD-53005.1 0.05 0.72 0.32 0.58 0.83 0.01 0.00 0.01 0.29 0.00 AD-53006.1 0.21 0.82 0.66 0.77 1.03 0.01 0.00 0.02 0.07 0.02 AD-53007.1 0.12 0.76 0.55 0.73 0.74 0.01 0.00 0.00 0.08 0.20 AD-53008.1 0.07 0.68 0.28 0.36 0.84 0.00 0.02 0.01 0.05 0.03 AD-53009.1 0.10 0.61 0.48 0.60 0.91 0.00 0.02 0.01 0.01 0.06 AD-53010.1 0.05 0.58 0.47 0.54 0.84 0.00 0.02 0.00 0.02 0.03 AD-53011.1 0.07 0.65 0.29 0.34 0.84 0.00 0.03 0.07 0.01 0.04 AD-53012.1 0.06 0.55 0.36 0.45 0.70 0.00 0.03 0.02 0.02 0.00 AD-53013.1 0.11 0.85 0.59 0.70 1.01 0.00 0.00 0.03 0.03 0.02 AD-53014.1 0.16 0.78 0.61 0.78 1.11 0.00 0.02 0.01 0.05 0.00 AD-53015.1 0.03 0.35 0.25 0.37 0.46 0.01 0.01 0.01 0.00 0.01 AD-53016.1 0.03 0.56 0.40 0.58 1.01 0.00 0.01 0.02 0.06 0.09 AD-53017.1 0.07 0.71 0.64 0.78 0.98 0.00 0.01 0.01 0.05 0.00 AD-53018.1 0.30 0.96 0.75 0.97 1.14 0.00 0.02 0.02 0.03 0.05 AD-53019.1 0.27 0.99 0.77 1.05 1.31 0.00 0.01 0.01 0.04 0.00 AD-53020.1 0.04 0.64 0.32 0.45 0.69 0.00 0.00 0.03 0.02 0.03 AD-53021.1 0.04 0.68 0.36 0.48 0.70 0.01 0.01 0.02 0.07 0.00 AD-53022.1 0.05 0.76 0.36 0.59 1.04 0.01 0.01 0.02 0.03 0.06 AD-53023.1 0.10 0.83 0.69 0.84 0.97 0.01 0.01 0.06 0.02 0.01 AD-53024.1 0.09 0.44 0.23 0.23 0.44 0.00 0.00 0.03 0.01 0.02 AD-53025.1 0.09 0.87 0.58 0.80 1.09 0.00 0.03 0.01 0.04 0.04 AD-53026.1 0.05 0.60 0.35 0.46 0.77 0.01 0.01 0.02 0.05 0.03 AD-53027.1 0.02 0.32 0.26 0.30 0.45 0.00 0.01 0.02 0.03 0.02 AD-53028.1 0.19 0.82 0.77 0.95 1.04 0.01 0.04 0.05 0.01 0.03 AD-53029.1 0.02 0.52 0.32 0.41 0.72 0.00 0.00 0.01 0.02 0.07 AD-53030.1 0.09 0.42 0.15 0.16 0.46 0.00 0.00 0.00 0.00 0.02 AD-53031.1 0.12 0.79 0.63 0.73 1.04 0.02 0.05 0.02 0.04 0.03 AD-53032.1 0.12 0.71 0.41 0.59 0.90 0.01 0.00 0.02 0.04 0.00 AD-53033.1 0.02 0.48 0.20 0.21 0.51 0.00 0.02 0.02 0.01 0.00 AD-53034.1 0.04 0.52 0.31 0.36 0.71 0.00 0.01 0.07 0.02 0.01 AD-53035.1 0.02 0.63 0.34 0.50 0.85 0.00 0.02 0.03 0.00 0.03 AD-53036.1 0.10 0.57 0.31 0.35 0.65 0.01 0.01 0.03 0.03 0.01 AD-53037.1 0.08 0.47 0.27 0.36 0.60 0.00 0.02 0.01 0.03 0.01 AD-53038.1 0.05 0.85 0.48 0.63 1.08 0.00 0.05 0.00 0.02 0.05 AD-53039.1 0.08 0.82 0.45 0.64 0.97 0.00 0.01 0.01 0.03 0.00 AD-53040.1 0.05 0.79 0.46 0.62 0.97 0.01 0.01 0.01 0.05 0.06 AD-53041.1 0.06 0.72 0.59 0.61 0.86 0.00 0.01 0.05 0.06 0.03 AD-53042.1 0.08 0.85 0.30 0.35 0.81 0.01 0.00 0.00 0.03 0.03 AD-53043.1 0.63 1.00 0.92 1.04 1.07 0.03 0.00 0.06 0.03 0.07 AD-53044.1 0.05 0.91 0.35 0.61 0.97 0.01 0.01 0.01 0.04 0.02 AD-53045.1 0.20 1.00 0.85 1.00 0.98 0.00 0.03 0.04 0.01 0.04 AD-53046.1 0.07 0.70 0.44 0.62 1.12 0.00 0.01 0.03 0.00 0.09 AD-53059.1 0.35 1.04 0.75 0.85 0.86 0.01 0.01 0.03 0.02 0.04 AD-53060.1 0.34 0.85 0.72 0.96 0.82 0.00 0.01 0.02 0.01 0.02 AD-53061.1 0.17 0.94 0.36 0.37 0.59 0.00 0.00 0.02 0.00 0.02 AD-53062.1 0.09 0.76 0.43 0.47 0.69 0.01 0.01 0.01 0.03 0.01 AD-53063.1 0.06 0.48 0.18 0.16 0.25 0.00 0.01 0.01 0.01 0.02 AD-53064.1 0.07 0.59 0.22 0.22 0.48 0.01 0.02 0.01 0.02 0.06 AD-53065.1 0.08 0.97 0.45 0.39 0.64 0.01 0.01 0.02 0.01 0.01 AD-53066.1 0.12 0.99 0.73 0.67 0.88 0.01 0.03 0.01 0.01 0.05 AD-53067.1 0.12 1.08 0.59 0.60 0.79 0.00 0.12 0.01 0.01 0.03 AD-53068.1 0.09 0.98 0.46 0.59 0.83 0.00 0.03 0.04 0.07 0.05 AD-53069.1 0.04 0.69 0.35 0.43 0.59 0.00 0.01 0.01 0.04 0.01 AD-53070.1 0.17 1.12 0.88 0.83 0.98 0.00 0.01 0.04 0.00 0.01 AD-53071.1 0.07 0.70 0.23 0.23 0.43 0.00 0.00 0.02 0.00 0.01 AD-53072.1 0.10 0.90 0.49 0.48 0.75 0.01 0.05 0.00 0.01 0.02 AD-53073.1 0.07 0.63 0.27 0.30 0.43 0.00 0.00 0.01 0.01 0.00 AD-53074.1 0.07 0.88 0.46 0.49 0.62 0.01 0.08 0.01 0.06 0.03 AD-53075.1 0.05 0.76 0.29 0.35 0.50 0.01 0.01 0.00 0.02 0.03 AD-53076.1 0.09 0.80 0.31 0.40 0.54 0.01 0.01 0.02 0.05 0.02 AD-53077.1 0.07 0.96 0.29 0.28 0.49 0.00 0.03 0.00 0.01 0.01 AD-53078.1 0.16 0.95 0.51 0.51 0.70 0.00 0.04 0.01 0.01 0.06 AD-53079.1 0.08 0.96 0.59 0.67 0.83 0.00 0.02 0.01 0.03 0.01 AD-53080.1 0.04 0.63 0.20 0.22 0.43 0.00 0.01 0.00 0.01 0.01 AD-53081.1 0.16 1.02 0.63 0.75 0.87 0.00 0.09 0.00 0.02 0.05 AD-53082.1 0.06 0.94 0.50 0.52 0.66 0.01 0.06 0.02 0.03 0.03 AD-53083.1 0.14 0.87 0.48 0.50 0.80 0.01 0.02 0.04 0.06 0.01 AD-53084.1 0.12 0.95 0.50 0.47 0.72 0.01 0.03 0.04 0.00 0.00 AD-53085.1 0.27 1.02 0.68 0.81 0.99 0.01 0.01 0.01 0.05 0.02 AD-53086.1 0.05 0.60 0.26 0.25 0.48 0.00 0.01 0.03 0.00 0.01 AD-53087.1 0.05 0.56 0.32 0.39 0.53 0.00 0.01 0.01 0.03 0.02 AD-53088.1 0.09 0.89 0.53 0.69 0.87 0.00 0.01 0.02 0.04 0.02 AD-53089.1 0.29 0.97 0.58 0.57 0.78 0.01 0.00 0.02 0.02 0.02 AD-53090.1 0.13 0.86 0.56 0.55 0.73 0.00 0.01 0.01 0.03 0.00 AD-53091.1 0.12 0.82 0.27 0.35 0.66 0.00 0.03 0.03 0.01 0.07 AD-53092.1 0.05 0.66 0.26 0.29 0.42 0.00 0.01 0.02 0.04 0.02 AD-53093.1 0.08 0.68 0.36 0.44 0.55 0.00 0.02 0.03 0.04 0.10 AD-53094.1 0.32 1.00 1.05 0.92 1.11 0.02 0.01 0.01 0.00 0.03 AD-53095.1 0.14 0.77 0.29 0.29 0.49 0.00 0.02 0.00 0.01 0.01 AD-53096.1 0.30 0.96 0.61 0.57 0.73 0.03 0.01 0.02 0.02 0.01 AD-53097.1 0.37 0.97 0.67 0.82 0.86 0.01 0.01 0.01 0.02 0.01 AD-53098.1 0.06 0.65 0.22 0.30 0.43 0.00 0.03 0.03 0.00 0.01 AD-53099.1 0.34 0.99 0.61 0.81 0.91 0.00 0.00 0.04 0.02 0.06 AD-53100.1 0.31 1.04 0.95 1.03 1.00 0.02 0.01 0.06 0.02 0.17 AD-53101.1 0.46 0.93 0.63 0.69 0.78 0.00 0.01 0.04 0.03 0.04 AD-53102.1 0.23 0.80 0.60 0.55 0.66 0.00 0.03 0.01 0.02 0.03 AD-53103.1 0.05 0.61 0.27 0.32 0.50 0.01 0.02 0.00 0.01 0.00 AD-53104.1 0.13 0.80 0.64 0.68 0.77 0.00 0.02 0.03 0.01 0.05 AD-53105.1 0.15 0.77 0.43 0.65 0.77 0.01 0.03 0.02 0.02 0.05 AD-53106.1 0.16 0.87 0.72 0.70 0.83 0.01 0.02 0.00 0.00 0.04 AD-53107.1 0.19 0.95 0.62 0.65 0.90 0.00 0.02 0.01 0.03 0.04 AD-53108.1 0.22 0.94 0.60 0.68 0.81 0.00 0.01 0.00 0.03 0.04 AD-53109.1 0.16 1.01 0.82 0.78 0.96 0.01 0.08 0.04 0.01 0.07 AD-53110.1 0.10 0.86 0.79 0.77 0.94 0.00 0.05 0.03 0.01 0.05 AD-53111.1 0.22 0.78 0.94 0.85 1.04 0.01 0.01 0.01 0.01 0.07 AD-53112.1 0.09 0.96 0.64 0.65 0.86 0.01 0.02 0.07 0.07 0.00 AD-53113.1 0.10 0.97 0.71 0.77 0.88 0.01 0.05 0.01 0.02 0.01 AD-53114.1 0.19 0.83 0.48 0.52 0.66 0.01 0.01 0.02 0.01 0.00 AD-53115.1 0.10 0.59 0.42 0.44 0.66 0.01 0.03 0.04 0.00 0.02 AD-53116.1 0.11 0.87 0.82 0.85 0.95 0.00 0.05 0.05 0.05 0.05 AD-53117.1 0.52 0.64 1.21 1.00 1.08 0.01 0.03 0.09 0.04 0.07 AD-53118.1 0.19 1.04 0.60 0.72 0.94 0.00 0.07 0.02 0.05 0.06 AD-53119.1 0.06 0.77 0.44 0.47 0.64 0.01 0.03 0.00 0.01 0.01 AD-53120.1 0.10 0.97 0.78 0.89 1.01 0.01 0.04 0.05 0.01 0.04 AD-53121.1 0.23 0.80 0.58 0.69 0.90 0.01 0.02 0.04 0.02 0.06 AD-53122.1 0.09 0.80 0.90 0.94 1.09 0.01 0.07 0.02 0.04 0.10 AD-53123.1 0.27 0.74 0.95 0.93 0.97 0.00 0.01 0.03 0.01 0.08 AD-53124.1 0.08 0.81 0.33 0.34 0.61 0.01 0.02 0.00 0.01 0.01 AD-53125.1 0.08 0.82 0.34 0.38 0.58 0.00 0.02 0.00 0.01 0.07 AD-53126.1 0.15 0.95 0.70 0.86 1.06 0.01 0.04 0.05 0.02 0.00 AD-53127.1 0.21 0.81 0.62 0.75 0.91 0.02 0.04 0.01 0.03 0.00 AD-53128.1 0.08 0.79 0.80 1.14 1.09 0.00 0.06 0.04 0.01 0.03 AD-53129.1 0.48 0.78 1.05 1.00 1.10 0.00 0.01 0.06 0.01 0.03 AD-53130.1 0.25 1.08 0.63 0.72 0.88 0.01 0.02 0.00 0.01 0.00 AD-53131.1 0.14 0.96 0.54 0.57 0.81 0.02 0.02 0.05 0.01 0.04 AD-53132.1 0.03 0.54 0.24 0.27 0.49 0.00 0.02 0.02 0.00 0.01 AD-53133.1 0.12 0.76 0.50 0.67 0.93 0.00 0.03 0.01 0.01 0.06 AD-53134.1 0.28 0.86 1.14 0.81 0.97 0.01 0.04 0.05 0.02 0.04 AD-53135.1 0.47 0.74 1.03 0.94 1.09 0.01 0.03 0.04 0.07 0.04 AD-53136.1 0.09 0.99 0.64 0.69 0.94 0.01 0.05 0.01 0.05 0.02 AD-53137.1 0.08 0.75 0.39 0.39 0.59 0.01 0.03 0.00 0.00 0.00 AD-53138.1 0.04 0.71 0.33 0.34 0.60 0.00 0.02 0.00 0.03 0.00 AD-53139.1 0.11 0.76 0.55 0.66 0.84 0.01 0.01 0.06 0.01 0.02 AD-53140.1 0.09 0.71 0.64 0.71 0.86 0.00 0.04 0.01 0.02 0.02 AD-53141.1 0.24 1.09 0.77 0.91 0.93 0.00 0.01 0.00 0.06 0.00 AD-53142.1 0.13 0.95 0.55 0.70 0.82 0.01 0.03 0.03 0.04 0.02 AD-53143.1 0.13 0.91 0.67 0.83 0.94 0.01 0.00 0.03 0.03 0.07 AD-53144.1 0.10 0.72 0.54 0.69 0.84 0.01 0.03 0.01 0.03 0.00 AD-53145.1 0.08 0.72 0.70 0.78 0.88 0.01 0.03 0.01 0.08 0.02 AD-53146.1 0.83 1.07 0.85 0.96 0.98 0.01 0.06 0.00 0.05 0.00 AD-53147.1 0.08 0.56 0.27 0.34 0.47 0.00 0.01 0.01 0.01 0.01 AD-53148.1 0.06 0.81 0.61 0.68 0.74 0.01 0.00 0.03 0.06 0.05 AD-53149.1 0.23 0.86 0.71 0.83 0.92 0.01 0.02 0.06 0.02 0.03 AD-53150.1 0.41 0.70 1.03 1.09 1.03 0.03 0.06 0.03 0.04 0.01

TABLE 12 Dose response screen results for ANGPTL3 GalNac-conjugated dsRNA sequences A subset of active siRNAs from the single dose screen (refer to data in Table 11) was tested in a dose response experiment by free uptake in PCH cells. A subset of these active siRNAs was also tested in dose response in Hep3B cells by transfection. IC₅₀ (nM) Free uptake Transfection (RNAiMax) AD-53063.1 1.60 0.03 AD-53001.1 2.27 0.01 AD-53015.1 2.90 0.02 AD-52953.1 2.94 0.03 AD-52986.1 3.30 0.03 AD-53024.1 3.42 0.02 AD-53033.1 3.42 0.02 AD-53027.1 3.84 0.01 AD-53030.1 3.90 0.03 AD-53080.1 4.08 0.04 AD-53073.1 4.20 0.05 AD-52965.1 4.63 ND AD-53092.1 5.37 ND AD-53132.1 5.54 ND AD-52983.1 5.55 ND AD-52954.1 5.67 ND AD-52961.1 6.37 ND AD-52994.1 6.43 ND AD-53098.1 6.58 ND AD-52970.1 6.71 ND AD-53075.1 6.74 ND AD-53086.1 7.08 ND AD-52971.1 7.50 ND AD-53064.1 8.33 ND AD-53147.1 8.34 ND AD-52969.1 8.86 ND AD-53077.1 8.98 ND AD-52981.1 9.44 ND AD-52977.1 10.45 ND AD-53071.1 11.19 ND AD-52960.1 13.03 ND AD-53095.1 21.31 ND AD-53103.1 21.92 ND

TABLE 13 Results of single dose screen using sequences listed in Table 10. 10 0.1 0.025 STDEV STDEV STDEV Duplex nM nM nM 10 nM 0.1 nM 0.025 nM AD-52719.1 0.01 0.60 0.35 0.000 0.093 0.002 AD-52717.1 0.02 0.31 0.32 0.001 0.014 0.008 AD-52713.1 0.02 0.37 0.36 0.001 0.011 0.007 AD-52711.1 0.03 0.22 0.23 0.005 0.011 0.009 AD-52718.1 0.03 0.31 0.39 0.000 0.025 0.023 AD-52687.1 0.03 0.37 0.38 0.005 0.020 0.002 AD-52699.1 0.03 0.25 0.21 0.002 0.011 0.002 AD-52679.1 0.03 0.51 0.24 0.345 0.008 AD-52689.1 0.03 0.44 0.42 0.000 0.039 0.002 AD-52700.1 0.03 0.56 0.57 0.005 0.044 0.020 AD-52637.1 0.04 0.27 0.23 0.001 0.003 0.005 AD-52730.1 0.04 0.61 0.59 0.005 0.053 0.014 AD-52725.1 0.04 0.62 0.61 0.002 0.027 0.012 AD-52688.1 0.04 0.23 0.20 0.006 0.012 0.011 AD-52661.1 0.04 0.61 0.25 0.001 0.449 0.009 AD-52667.1 0.04 0.28 0.22 0.004 0.018 0.013 AD-52665.1 0.04 0.43 0.48 0.007 0.019 0.009 AD-52638.1 0.04 0.28 0.25 0.000 0.016 0.027 AD-52724.1 0.05 0.86 0.76 0.001 0.055 0.011 AD-52705.1 0.05 0.74 0.65 0.004 0.022 0.016 AD-52708.1 0.05 0.53 0.52 0.001 0.034 0.013 AD-52659.1 0.05 0.56 0.48 0.000 0.000 0.033 AD-52678.1 0.05 0.53 0.53 0.002 0.034 0.000 AD-52670.1 0.05 0.35 0.33 0.002 0.009 0.003 AD-52695.1 0.05 0.63 0.67 0.001 0.012 0.013 AD-52704.1 0.05 0.55 0.53 0.002 0.005 0.034 AD-52683.1 0.05 0.36 0.28 0.002 0.021 0.011 AD-52673.1 0.05 0.22 0.19 0.023 0.010 0.002 AD-52721.1 0.05 0.60 0.53 0.003 0.006 0.029 AD-52710.1 0.05 0.56 0.40 0.007 0.073 0.000 AD-52714.1 0.05 0.40 0.51 0.000 0.016 0.003 AD-52686.1 0.05 0.57 0.60 0.003 0.014 0.000 AD-52645.1 0.05 0.62 0.59 0.004 0.030 0.003 AD-52662.1 0.05 0.55 0.52 0.002 0.030 0.008 AD-52720.1 0.05 0.50 0.46 0.003 0.007 0.011 AD-52654.1 0.05 0.29 0.36 0.008 0.037 0.014 AD-52680.1 0.06 0.48 0.41 0.001 0.019 0.026 AD-52723.1 0.06 0.84 0.76 0.001 0.041 0.004 AD-52726.1 0.06 0.72 0.66 0.003 0.028 0.016 AD-52701.1 0.06 0.67 0.39 0.001 0.003 0.002 AD-52694.1 0.06 0.68 0.59 0.004 0.040 0.012 AD-52685.1 0.06 0.30 0.25 0.002 0.013 0.016 AD-52728.1 0.06 0.80 0.79 0.005 0.043 0.015 AD-52676.1 0.06 0.68 0.67 0.002 0.023 0.029 AD-52639.1 0.06 0.47 0.45 0.000 0.005 0.007 AD-52722.1 0.06 0.81 0.93 0.005 0.004 0.027 AD-52682.1 0.06 0.87 0.73 0.009 0.038 0.014 AD-52660.1 0.07 0.69 0.68 0.002 0.014 0.017 AD-52709.1 0.07 0.89 0.82 0.001 0.013 0.020 AD-52643.1 0.07 0.27 0.24 0.006 0.016 0.012 AD-52696.1 0.07 0.53 0.46 0.003 0.026 0.007 AD-52657.1 0.08 0.60 0.58 0.008 0.030 0.006 AD-52706.1 0.08 0.84 0.78 0.001 0.021 0.019 AD-52653.1 0.08 0.41 0.45 0.057 0.004 0.029 AD-52656.1 0.08 0.65 0.50 0.004 0.022 0.012 AD-52693.1 0.09 0.61 0.62 0.007 0.021 0.018 AD-52692.1 0.09 0.54 0.52 0.023 0.018 0.033 AD-52674.1 0.10 0.79 0.64 0.001 0.008 0.028 AD-52648.1 0.10 0.67 0.53 0.002 0.013 0.028 AD-52651.1 0.10 0.84 0.73 0.000 0.000 0.007 AD-52641.1 0.10 0.62 0.50 0.004 0.172 0.002 AD-52707.1 0.10 0.92 0.81 0.001 0.018 0.032 AD-52671.1 0.11 0.87 0.84 0.005 0.034 0.025 AD-52650.1 0.12 0.88 0.94 0.007 0.013 0.041 AD-52642.1 0.12 0.90 0.76 0.015 0.022 0.004 AD-52675.1 0.13 0.94 0.89 0.001 0.018 0.044 AD-52647.1 0.13 0.80 0.79 0.031 0.008 0.023 AD-52716.1 0.14 0.61 0.69 0.010 0.060 0.013 AD-52649.1 0.14 0.31 0.29 0.136 0.020 0.006 AD-52677.1 0.16 1.01 0.72 0.059 0.040 0.007 AD-52697.1 0.16 0.86 0.77 0.012 0.021 0.015 AD-52715.1 0.17 0.90 0.89 0.005 0.009 0.022 AD-52691.1 0.18 0.93 0.88 0.004 0.036 0.017 AD-52698.1 0.20 0.97 0.87 0.010 0.028 0.000 AD-52672.1 0.20 0.70 0.66 0.170 0.014 0.019 AD-52712.1 0.29 0.92 0.90 0.007 0.036 0.004 AD-52690.1 0.30 0.95 0.85 0.115 0.032 0.004 AD-52640.1 0.30 1.04 0.91 0.018 0.046 0.013 AD-52684.1 0.31 0.90 0.94 0.014 0.018 0.014 AD-52666.1 0.32 1.04 0.91 0.013 0.005 0.004 AD-52703.1 0.32 1.02 0.96 0.016 0.015 0.005 AD-52729.1 0.33 1.02 0.87 0.032 0.020 0.008 AD-52668.1 0.35 0.94 0.90 0.029 0.046 0.026 AD-52681.1 0.57 1.00 0.99 0.003 0.034 0.039 AD-52702.1 0.72 1.02 0.92 0.658 0.060 0.014 AD-52727.1 0.73 1.03 0.91 0.004 0.065 0.027 AD-52663.1 0.78 1.05 0.96 0.027 0.010 0.005 AD-52669.1 0.91 0.91 0.94 0.004 0.049 0.032 AD-1955 0.95 0.84 0.95 0.005 0.021 0.019 AD-1955 0.97 1.07 1.03 0.000 0.021 0.015 AD-1955 1.01 1.08 1.01 0.035 0.011 0.005 mock 1.02 0.96 0.97 0.030 0.037 0.005 AD-1955 1.08 1.03 1.02 0.032 0.051 0.005 AD-52652.1 1.13 1.11 1.02 0.028 0.043 0.020 AD-52658.1 1.33 1.10 0.93 0.091 0.043 0.018 AD-52664.1 1.49 0.95 0.88 0.438 0.019 0.009 AD-52752.1 0.03 0.43 0.69 0.002 0.015 0.017 AD-52741.1 0.03 0.56 0.86 0.001 0.044 0.021 AD-52804.1 0.03 0.49 0.89 0.001 0.002 0.017 AD-52764.1 0.03 0.54 0.79 0.005 0.016 0.078 AD-52770.1 0.03 0.58 0.78 0.000 0.006 0.027 AD-52735.1 0.03 0.31 0.46 0.003 0.031 0.009 AD-52810.1 0.03 0.67 0.86 0.001 0.013 0.025 AD-52759.1 0.03 0.54 0.79 0.000 0.018 0.023 AD-52736.1 0.03 0.51 0.60 0.004 0.012 0.023 AD-52775.1 0.03 0.54 0.73 0.005 0.024 0.022 AD-52758.1 0.03 0.57 0.78 0.001 0.014 0.050 AD-52743.1 0.03 0.45 0.67 0.002 0.018 0.033 AD-52747.1 0.04 0.57 0.84 0.002 0.061 0.058 AD-52819.1 0.04 0.26 0.45 0.005 0.001 0.022 AD-52765.1 0.04 0.68 0.83 0.000 0.013 0.053 AD-52754.1 0.04 0.76 1.00 0.000 0.007 0.015 AD-52787.1 0.05 0.55 0.68 0.001 0.043 0.060 AD-52791.1 0.05 0.70 0.91 0.001 0.014 0.084 AD-52811.1 0.05 0.73 0.84 0.002 0.014 0.058 AD-52817.1 0.05 0.77 0.92 0.003 0.011 0.031 AD-52745.1 0.06 0.62 0.77 0.007 0.021 0.000 AD-52749.1 0.06 0.63 0.88 0.005 0.037 0.043 AD-52740.1 0.06 0.83 0.94 0.007 0.012 0.051 AD-52796.1 0.06 0.72 0.92 0.003 0.021 0.054 AD-52820.1 0.06 0.90 0.87 0.001 0.026 0.064 AD-52809.1 0.06 0.76 0.90 0.001 0.037 0.027 AD-52760.1 0.06 0.81 0.97 0.001 0.056 0.047 AD-52767.1 0.07 0.55 0.55 0.001 0.016 0.013 AD-52734.1 0.07 0.61 0.64 0.004 0.003 0.003 AD-52794.1 0.07 0.94 0.87 0.007 0.014 0.051 AD-52797.1 0.07 0.69 0.87 0.004 0.000 0.038 AD-52737.1 0.08 0.70 0.84 0.004 0.031 0.012 AD-52812.1 0.08 0.75 0.88 0.004 0.000 0.056 AD-52748.1 0.08 0.70 0.89 0.001 0.010 0.009 AD-52782.1 0.08 0.68 0.78 0.004 0.023 0.011 AD-52816.1 0.08 0.71 0.88 0.003 0.042 0.060 AD-52763.1 0.08 0.68 0.77 0.002 0.013 0.026 AD-52788.1 0.08 0.89 1.00 0.004 0.017 0.034 AD-52762.1 0.08 0.78 0.91 0.007 0.046 0.009 AD-52785.1 0.08 0.88 0.95 0.002 0.004 0.019 AD-52800.1 0.09 0.82 0.94 0.001 0.040 0.005 AD-52792.1 0.09 0.93 0.94 0.002 0.018 0.037 AD-52784.1 0.10 0.84 0.92 0.000 0.066 0.032 AD-52746.1 0.10 0.82 0.93 0.002 0.060 0.059 AD-52814.1 0.10 0.85 0.88 0.002 0.042 0.013 AD-52751.1 0.10 0.88 0.98 0.005 0.030 0.067 AD-52786.1 0.10 0.81 0.81 0.006 0.028 0.048 AD-52755.1 0.10 0.93 0.99 0.003 0.032 0.048 AD-52808.1 0.11 0.98 0.92 0.000 0.038 0.032 AD-52815.1 0.11 0.96 0.96 0.002 0.009 0.000 AD-52805.1 0.11 0.79 0.86 0.003 0.050 0.008 AD-52777.1 0.11 0.88 0.94 0.001 0.065 0.000 AD-52756.1 0.11 0.92 0.91 0.003 0.032 0.004 AD-52733.1 0.12 0.66 0.65 0.005 0.071 0.022 AD-52739.1 0.13 0.83 0.95 0.002 0.008 0.061 AD-52780.1 0.13 0.70 0.67 0.012 0.021 0.059 AD-52798.1 0.13 0.64 0.97 0.001 0.006 0.038 AD-52776.1 0.14 0.97 0.94 0.011 0.029 0.023 AD-52753.1 0.15 0.88 1.09 0.001 0.048 0.005 AD-52778.1 0.16 0.76 0.69 0.003 0.067 0.003 AD-52744.1 0.16 0.90 0.91 0.002 0.000 0.049 AD-52750.1 0.16 0.87 1.01 0.000 0.060 0.055 AD-52774.1 0.17 0.71 0.89 0.002 0.010 0.017 AD-52803.1 0.18 0.87 0.92 0.015 0.026 0.040 AD-52821.1 0.18 0.86 0.87 0.005 0.046 0.055 AD-52781.1 0.18 0.78 0.66 0.008 0.000 0.023 AD-52779.1 0.20 0.83 0.66 0.002 0.024 0.016 AD-52793.1 0.20 0.74 0.88 0.010 0.025 0.069 AD-52799.1 0.20 0.75 1.01 0.005 0.018 0.010 AD-52761.1 0.22 0.83 0.92 0.000 0.024 0.023 AD-52768.1 0.22 0.96 0.97 0.001 ND 0.028 AD-52757.1 0.23 1.02 0.95 0.018 0.040 0.042 AD-52806.1 0.24 0.96 0.87 0.011 0.084 0.055 AD-52771.1 0.25 0.92 0.98 0.010 0.018 0.048 AD-52802.1 0.30 0.95 1.00 0.010 0.019 0.005 AD-52731.1 0.30 0.85 0.75 0.001 0.067 0.022 AD-52813.1 0.30 1.07 0.98 0.001 0.109 0.014 AD-52742.1 0.31 0.95 1.03 0.005 0.028 0.056 AD-52766.1 0.35 0.97 1.00 0.010 0.024 0.044 AD-52732.1 0.41 0.79 0.73 0.004 0.016 0.039 AD-52773.1 0.43 0.99 0.92 0.004 0.029 0.022 AD-52772.1 0.43 1.00 1.02 0.006 0.000 0.065 AD-52822.1 0.44 0.68 0.81 0.004 0.010 0.016 AD-52783.1 0.45 0.66 0.76 0.009 0.036 0.019 AD-52789.1 0.50 0.68 0.78 0.010 0.053 0.004 AD-52795.1 0.50 0.82 0.69 0.000 0.080 0.054 AD-52801.1 0.54 0.70 0.79 0.018 0.038 0.035 AD-52807.1 0.57 0.76 0.93 0.006 0.011 0.032 AD-52769.1 0.76 0.97 0.92 0.015 0.085 0.045 AD-1955 0.90 0.96 1.04 0.018 0.165 0.010 AD-52818.1 0.92 1.03 0.92 0.009 0.010 0.063 AD-1955 1.01 0.90 0.96 0.005 0.031 0.019 AD-1955 1.05 1.09 1.00 0.046 0.085 0.005 AD-1955 1.05 1.07 1.00 0.010 0.031 0.039 mock 1.20 0.98 0.92 0.000 0.014 0.005 mock 1.25 0.99 1.00 0.006 0.005 0.034

TABLE 14 Results of a dose response screen using a subset of sequences from Table 13. A subset of active ANGPTL3 siRNAs from Table 10 were tested by transfection in Hep3B cells in dose response screens. Duplex IC50 (nM) AD-52819.1 0.0036 AD-52667.1 0.0037 AD-52638.1 0.0048 AD-52673.1 0.0049 AD-52711.1 0.0050 AD-52661.1 0.0054 AD-52654.1 0.0058 AD-52637.1 0.0058 AD-52643.1 0.0060 AD-52685.1 0.0062 AD-52670.1 0.0064 AD-52679.1 0.0064 AD-52649.1 0.0066 AD-52683.1 0.0069 AD-52688.1 0.0071 AD-52717.1 0.0072 AD-52699.1 0.0073 AD-52714.1 0.0086 AD-52718.1 0.0088 AD-52735.1 0.0093 AD-52653.1 0.0102 AD-52687.1 0.0109 AD-52680.1 0.0120 AD-52713.1 0.0133 AD-52720.1 0.0143 AD-52639.1 0.0161 AD-52696.1 0.0163 AD-52662.1 0.0179 AD-52659.1 0.0180 AD-52710.1 0.0195 AD-52689.1 0.0216 AD-52787.1 0.0242 AD-52765.1 0.0318

TABLE 15 IDs of duplex pairs for which both an unconjuaged and a GalNac- conjugated version were synthesized and tested These duplexes have the same sequence and modification pattern. Unconjugated GalNac conjugated duplex ID duplex ID AD-52637.1 AD-52953.1 AD-52638.1 AD-52954.1 AD-52639.1 AD-52955.1 AD-52640.1 AD-52956.1 AD-52641.1 AD-52957.1 AD-52642.1 AD-52958.1 AD-52643.1 None None AD-52960.1 None AD-52961.1 AD-52645.1 AD-52962.1 AD-52647.1 AD-52963.1 AD-52648.1 AD-52964.1 AD-52649.1 AD-52965.1 AD-52650.1 AD-52966.1 AD-52651.1 AD-52967.1 AD-52652.1 AD-52968.1 AD-52653.1 AD-52969.1 AD-52654.1 AD-52970.1 None AD-52971.1 AD-52656.1 AD-52972.1 AD-52657.1 AD-52973.1 AD-52658.1 AD-52974.1 AD-52659.1 AD-52975.1 AD-52660.1 AD-52976.1 AD-52661.1 AD-52977.1 AD-52662.1 AD-52978.1 AD-52663.1 AD-52979.1 AD-52664.1 AD-52980.1 AD-52665.1 AD-52981.1 AD-52666.1 AD-52982.1 AD-52667.1 AD-52983.1 AD-52668.1 AD-52984.1 AD-52669.1 AD-52985.1 AD-52670.1 AD-52986.1 AD-52671.1 AD-52987.1 AD-52672.1 AD-52988.1 AD-52673.1 AD-52989.1 AD-52674.1 AD-52990.1 AD-52675.1 AD-52991.1 AD-52676.1 AD-52992.1 AD-52677.1 AD-52993.1 AD-52678.1 AD-52994.1 AD-52679.1 AD-52995.1 AD-52680.1 AD-52996.1 AD-52681.1 AD-52997.1 AD-52682.1 AD-52998.1 AD-52683.1 AD-52999.1 AD-52684.1 AD-53000.1 AD-52685.1 AD-53001.1 AD-52686.1 AD-53002.1 AD-52687.1 AD-53003.1 AD-52688.1 AD-53004.1 AD-52689.1 AD-53005.1 AD-52690.1 AD-53006.1 AD-52691.1 AD-53007.1 AD-52692.1 AD-53008.1 AD-52693.1 AD-53009.1 AD-52694.1 AD-53010.1 AD-52695.1 AD-53011.1 AD-52696.1 AD-53012.1 AD-52697.1 AD-53013.1 AD-52698.1 AD-53014.1 AD-52699.1 AD-53015.1 AD-52700.1 AD-53016.1 AD-52701.1 AD-53017.1 AD-52702.1 AD-53018.1 AD-52703.1 AD-53019.1 AD-52704.1 AD-53020.1 AD-52705.1 AD-53021.1 AD-52706.1 AD-53022.1 AD-52707.1 AD-53023.1 AD-52708.1 AD-53024.1 AD-52709.1 AD-53025.1 AD-52710.1 AD-53026.1 AD-52711.1 AD-53027.1 AD-52712.1 AD-53028.1 AD-52713.1 AD-53029.1 AD-52714.1 AD-53030.1 AD-52715.1 AD-53031.1 AD-52716.1 AD-53032.1 AD-52717.1 AD-53033.1 AD-52718.1 AD-53034.1 AD-52719.1 AD-53035.1 AD-52720.1 AD-53036.1 AD-52721.1 AD-53037.1 AD-52722.1 AD-53038.1 AD-52723.1 AD-53039.1 AD-52724.1 AD-53040.1 AD-52725.1 AD-53041.1 AD-52726.1 AD-53042.1 AD-52727.1 AD-53043.1 AD-52728.1 AD-53044.1 AD-52729.1 AD-53045.1 AD-52730.1 AD-53046.1 AD-52731.1 AD-53059.1 AD-52732.1 AD-53060.1 AD-52733.1 AD-53061.1 AD-52734.1 AD-53062.1 AD-52735.1 AD-53063.1 AD-52736.1 AD-53064.1 AD-52737.1 AD-53065.1 None AD-53066.1 AD-52739.1 AD-53067.1 AD-52740.1 AD-53068.1 AD-52741.1 AD-53069.1 AD-52742.1 AD-53070.1 AD-52743.1 AD-53071.1 AD-52744.1 AD-53072.1 AD-52745.1 AD-53073.1 AD-52746.1 AD-53074.1 AD-52747.1 AD-53075.1 AD-52748.1 AD-53076.1 AD-52749.1 AD-53077.1 AD-52750.1 AD-53078.1 AD-52751.1 AD-53079.1 AD-52752.1 AD-53080.1 AD-52753.1 AD-53081.1 AD-52754.1 AD-53082.1 AD-52755.1 AD-53083.1 AD-52756.1 AD-53084.1 AD-52757.1 AD-53085.1 AD-52758.1 AD-53086.1 AD-52759.1 AD-53087.1 AD-52760.1 AD-53088.1 AD-52761.1 AD-53089.1 AD-52762.1 AD-53090.1 AD-52763.1 AD-53091.1 AD-52764.1 AD-53092.1 AD-52765.1 AD-53093.1 AD-52766.1 AD-53094.1 AD-52767.1 AD-53095.1 AD-52768.1 AD-53096.1 AD-52769.1 AD-53097.1 AD-52770.1 AD-53098.1 AD-52771.1 AD-53099.1 AD-52772.1 AD-53100.1 AD-52773.1 AD-53101.1 AD-52774.1 AD-53102.1 AD-52775.1 AD-53103.1 AD-52776.1 AD-53104.1 AD-52777.1 AD-53105.1 AD-52778.1 AD-53106.1 AD-52779.1 AD-53107.1 AD-52780.1 AD-53108.1 AD-52781.1 AD-53109.1 AD-52782.1 AD-53110.1 AD-52783.1 AD-53111.1 AD-52784.1 AD-53112.1 AD-52785.1 AD-53113.1 AD-52786.1 AD-53114.1 AD-52787.1 AD-53115.1 AD-52788.1 AD-53116.1 AD-52789.1 AD-53117.1 None AD-53118.1 AD-52791.1 AD-53119.1 AD-52792.1 AD-53120.1 AD-52793.1 AD-53121.1 AD-52794.1 AD-53122.1 AD-52795.1 AD-53123.1 AD-52796.1 AD-53124.1 AD-52797.1 AD-53125.1 AD-52798.1 AD-53126.1 AD-52799.1 AD-53127.1 AD-52800.1 AD-53128.1 AD-52801.1 AD-53129.1 AD-52802.1 AD-53130.1 AD-52803.1 AD-53131.1 AD-52804.1 AD-53132.1 AD-52805.1 AD-53133.1 AD-52806.1 AD-53134.1 AD-52807.1 AD-53135.1 AD-52808.1 AD-53136.1 AD-52809.1 AD-53137.1 AD-52810.1 AD-53138.1 AD-52811.1 AD-53139.1 AD-52812.1 AD-53140.1 AD-52813.1 AD-53141.1 AD-52814.1 AD-53142.1 AD-52815.1 AD-53143.1 AD-52816.1 AD-53144.1 AD-52817.1 AD-53145.1 AD-52818.1 AD-53146.1 AD-52819.1 AD-53147.1 AD-52820.1 AD-53148.1 AD-52821.1 AD-53149.1 AD-52822.1 AD-53150.1 In Vivo Tests

Example 3

Test Articles

In vivo experiments were conducted using dsRNA sequences of the invention. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). The dsRNA sequence used as a negative control was luciferase-conjugated AD-48399B1 (“Luc”, sense sequence: CfaCfuUfaCfgCfuGfaGfuAfcUfuCfgAfL96 (SEQ ID NO: 1728), antisense sequence: uCfgAfaGfuAfcUfcAfgCfgUfaAfgUfgsAfsu (SEQ ID NO: 1729)). Also used as a negative control was GalNal-conjugated AD-1955 containing alternating 2′-methyl and 2′ fluoro modifications.

Experimental Procedure

The dsRNA sequences were tested in C57BL/6 (WT) and ob/ob mice. WT mice received five daily doses of dsRNAs in PBS, Luc at 20 mg/kg, or ANG at 5 or 20 mg/kg; and ob/ob mice received five daily doses of NPLs formulated with Luc at 20 mg/kg or ANG at 20 mg/kg. All test articles were administered by subcutaneous injection according to the procedure shown in FIG. 1. Specifically, five daily doses of the test articles were administered on five consecutive days (day 0, 1, 2, 3 and 4), and blood samples were collected 5, 3 or 1 day prior to administration, as well as on days 0, 1, 2, 3, 4, 7, 9, 11, 15, 18, 21, 25, 30, 37, 45 and 50 post-administration. The collected blood samples were used to measure the expression of ANGPTL3 protein using an ELISA assay. Levels of serum triglycerides (TGs), low density lipoprotein cholesterol (LDLc), high density lipoprotein cholesterol (HDLc) and total cholesterol (TC) were also measured using an Olympus Analyzer.

Results

Shown in FIG. 2, Panel A, are levels of murine ANGPTL3 (mANGPTL3, protein measured in WT mice after administration of control or ANG at 5 or 20 mg/kg. Also shown in FIG. 2, Panel B are levels of mANGPTL3 protein measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that, for both WT and ob/ob mice, administration of ANG results in decreased levels of mANGPTL3 protein, as compared to controls.

Shown in FIG. 3, Panel A, are levels of LDL-c measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 3, Panel B are levels of LDL-c measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of LDL-c, particularly in ob/ob mice, as compared to controls.

Shown in FIG. 4, Panel A, are levels of triglycerides measured in WT mice after administration of control or ANG at 20 mg/kg. Shown in FIG. 4, Panel B are levels of triglycerides measured in ob/ob mice after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes decreased levels of tryglycerides, particularly, in ob/ob mice, as compared to controls.

Shown in FIG. 5, Panel A and B are levels of total cholesterol (TC) measured in WT and ob/ob mice, respectively, after administration of control or ANG at 20 mg/kg. The data indicates that administration of ANG causes a moderate decrease in TC levels in ob/ob mice, but not in WT mice. Similarly, administration of ANG causes a moderate decrease in HDL-c levels in ob/ob mice, but not in WT mice, as is shown in the graphs in FIG. 6.

Example 4

Test Article

The effect of a single injection of dsRNA sequence of the invention on the level of ANGPTL3 protein was tested. The dsRNA sequence used in the experiments was GalNac-conjugated AD-52981 (“ANG”, sense sequence: AfcAfuAfuUfuGfAfUfcAfgUfcUfuUfuUfL96 (SEQ ID NO: 657); antisense sequence: aAfaAfaGfaCfuGfaucAfaAfuAfuGfusUfsg (SEQ ID NO: 842)). PBS was used as a negative control.

Experimental Procedure

The dsRNA sequences were tested in Human PCS Transgenic mouse characterized by liver-specific expression of full-length human PCSK9 gene. Human PCS transgenic mice were dosed with the AD-52981 or PBS using a single subcutaneous injection. The mice were divided into four groups, each group consisting of two males and two females. Each group received an injection of PBS or a 5 mg/kg, 20 mg/kg or 60 mg/kg dose of AD-52981. Blood samples were collected at day 1 and day 0 prior to dosing, and at 72 hours post dosing. ANGPTL3 protein levels were measured by ELISA and compared to levels at day 1 and day 0 prior to dosing.

Results

Shown in FIG. 7, are levels of murine ANGPTL3 protein (mANGPTL3) measured in Human PCS transgenic mice. The data shown is expressed relative to PBS control and represents an average for 2 males and 2 females in each group. Error bars represent standard deviation. The data indicates that administration of a single injection of AD-52981 reduces the levels of ANGPTL3 protein in the mice in a dose-dependent manner, with the dose of 60 mg/kg decreasing the levels of ANGPTL3 protein more than five-fold (see FIG. 7).

SEQUENCES >gi|41327750|ref|NM_014495.2| Homo sapiens angiopoietin-like 3 (ANGPTL3), mRNA SEQ ID NO: 1 TTCCAGAAGAAAACAGTTCCACGTTGCTTGAAATTGAAAATCAAGATAAAAATGTTCACAATTAAGCTCCT TCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGATCAAGACAATTCATCATTTGATTCTCTATCTC CAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGA CATGGTCTTAAAGACTTTGTCCATAAGACGAAGGGCCAAATTAATGACATATTTCAAAAACTCAACATATT TGATCAGTCTTTTTATGATCTATCGCTGCAAACCAGTGAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAA CTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATATGTCACTTGAACTCAACTCAAAACTTGAA AGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAATATTTAGAAGAGCAACTAACTAACTTAAT TCAAAATCAACCTGAAACTCCAGAACACCCAGAAGTAACTTCACTTAAAACTTTTGTAGAAAAACAAGATA ATAGCATCAAAGACCTTCTCCAGACCGTGGAAGACCAATATAAACAATTAAACCAACAGCATAGTCAAATA AAAGAAATAGAAAATCAGCTCAGAAGGACTAGTATTCAAGAACCCACAGAAATTTCTCTATCTTCCAAGCC AAGAGCACCAAGAACTACTCCCTTTCTTCAGTTGAATGAAATAAGAAATGTAAAACATGATGGCATTCCTG CTGAATGTACCACCATTTATAACAGAGGTGAACATACAAGTGGCATGTATGCCATCAGACCCAGCAACTCT CAAGTTTTTCATGTCTACTGTGATGTTATATCAGGTAGTCCATGGACATTAATTCAACATCGAATAGATGG ATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTTGGGAGGCTTGATGGAGAATTTTGGT TGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTATGTTTTACGAATTGAGTTGGAAGACTGG AAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAAACCAACTATACGCTACATCT AGTTGCGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTTGGTGTTTTCTACTTGGGATC ACAAAGCAAAAGGACACTTCAACTGTCCAGAGGGTTATTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGA GAAAACAACCTAAATGGTAAATATAACAAACCAAGAGCAAAATCTAAGCCAGAGAGGAGAAGAGGATTATC TTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTTGATCCATCCAACAGATTCAG AAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAATTTAAACATTAACCTCATTCCAAGTTAATGT GGTCTAATAATCTGGTATTAAATCCTTAAGAGAAAGCTTGAGAAATAGATTTTTTTTATCTTAAAGTCACT GTCTATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCGTTTACATTTCTCAATCAAAATTCT TATAATACTATTTGTTTTAAATTTTGTGATGTGGGAATCAATTTTAGATGGTCACAATCTAGATTATAATC AATAGGTGAACTTATTAAATAACTTTTCTAAATAAAAAATTTAGAGACTTTTATTTTAAAAGGCATCATAT GAGCTAATATCACAACTTTCCCAGTTTAAAAAACTAGTACTCTTGTTAAAACTCTAAACTTGACTAAATAC AGAGGACTGGTAATTGTACAGTTCTTAAATGTTGTAGTATTAATTTCAAAACTAAAAATCGTCAGCACAGA GTATGTGTAAAAATCTGTAATACAAATTTTTAAACTGATGCTTCATTTTGCTACAAAATAATTTGGAGTAA ATGTTTGATATGATTTATTTATGAAACCTAATGAAGCAGAATTAAATACTGTATTAAAATAAGTTCGCTGT CTTTAAACAAATGGAGATGACTACTAAGTCACATTGACTTTAACATGAGGTATCACTATACCTTATT >gi|297278846|ref|XM_001086114.2| PREDICTED: Macaca mulatta angiopoietin-like 3 (ANGPTL3), mRNA SEQ ID NO: 2 ATATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGTTGCTTGAAATTGAAAATC AGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATTGACCA AGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGTAAAAA TTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCCAAATT AATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGTGAAAT CAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAAGAATA TGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAGTGAAA TATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGAAACTCCAGAACATCCAGAAGTAACTTC ACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACAATATA AGCAATTAAACCAACAGCACAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTCAAGAA CCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAATGAAAT AAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATATAAGTG GCATGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTAAAACC TGTCTAAGGAGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAATATGGTTTCGGGAG GCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATCTAATTACGTTTTAC GAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACTTGGGAAATCACGAA ACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCGGAAAACAAAGATTT GGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTATTCAGGAGGCTGGT GGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAACAAAATCTAAGCCA GAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAATCAACCAAAATGTT GATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAATAAATTAAACATTA AACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGCTTGAGAAATAGATT TTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAAAGAATACCATTTAC ATTTCTCAATCAAAATTCCTACAACACTATTTGTTTTATATTTTGTGATGTGGGAATCAATTTTAGATGGT CGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAAACTTAGAGACTTTA ATTTTAAAAGTCATCATATGAGCTAATATCACAATTTTCCCAGTTTAAAAAACTAGTTTTCTTGTTAAAAC TCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTAATATTAATTTCAAA ACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACTGATGCCTCATTTTG CTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAGCAGGATTAAATACT GTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCACATTGACTTTAATAT GAGGTATCACTATACCTTA >gi|142388354|ref|NM_013913.3| Mus musculus angiopoietin-like 3 (Angptl3), mRNA SEQ ID NO: 3 CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTC CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC ATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTT GATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA GTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAA CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAA AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA AGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCC AAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC TCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTT GGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA AAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTG GCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCA CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGA AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA TCAGATATCACCGAATCAACGTAAACAAAATTTATC >gi|68163568|ref|NM_001025065.1| Rattus norvegicus angiopoietin-like 3 (Angptl3), mRNA SEQ ID NO: 4 GACGTTCCAAATTGCTTGAAATTGAATAATTGAAACAAAAATGCACACAATTAAGCTGCTCCTTTTTGTTG TTCCTCTAGTAATTTCGTCCAGAGTTGATCCAGACCTTTCGCCATTTGATTCTGTACCGTCAGAGCCAAAA TCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCCAATGGCCTCCTGCAGCTGGGTCATGGTCTTAA AGATTTTGTCCATAAGACAAAGGGACAAATTAATGACATATTTCAGAAGCTCAACATATTTGATCAGTGTT TTTATGACCTATCACTTCAAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAACCACATCTAAA CTACAAGTTAAAAACGAAGAGGTGAAGAATATGTCACTTGAACTGAACTCAAAGCTTGAAAGTCTACTGGA GGAGAAGATGGCGCTCCAACACAGAGTCAGGGCTTTGGAGGAACAGCTGACCAGCTTGGTTCAGAACCCGC CTGGGGCTCGGGAGCACCCAGAGGTAACGTCACTTAAAAGTTTTGTAGAACAGCAAGATAACAGCATAAGA GAACTCCTCCAGAGTGTGGAAGAACAATATAAACAACTAAGTCAACAGCACATTCAGATAAAAGAAATAGA AAATCAGCTCAGAAAGACTGGCATTCAAGAACCCACTGAAAATTCTCTTTATTCTAAACCAAGAGCACCAA GAACTACTCCCCCTCTTCATCTGAAGGAAGCAAAAAATATAGAACAAGATGATCTGCCTGCTGACTGCTCT GCCATTTATAACAGAGGTGAACATACAAGTGGCGTGTATACTATTAGACCAAGCAGCTCTCAAGTGTTTAA TGTCTACTGTGACACCCAATCAGGCACTCCACGGACATTAATTCAACACCGGAAAGATGGCTCTCAAAACT TCAACCAAACGTGGGAAAACTACGAAAAGGGTTTTGGGAGGCTTGATGGTAAAGTGATTTCCTTGCATCAC TCACTTATCTGTTGATTTAATAGTATTAGTTGGGTGTGTTGACACAGGCCTGAGACCATAGCGCTTTTGGG CAAGGGGGGAGGAGGAGCAGCAGGTGAATTGAAAGTTCAAGACCAGTCTGGGCCACACATTGATACTCCTT CTCGACATTAAGAATTATAAATTAAGCAGCAATTATAAAATGGGCTGTGGAAATGTAACAATAAGCAAAAG CAGACCCCAGTCTTCATAAAACTGATTGGTAAATATTATCCATGATAGCAACTGCAATGATCTCATTGTAC TTATCACTACTGCATGCCTGCAGTATGCTTGTTGAAACTTAATTCTATAGTTCATGGTTATCATAAGTCTT ATTAAGGAACATAGTATACGCCATTGGCTCTAGTGAGGGGCCATGCTACAAATGAGCTGCAAAGATAGCAG TATAGAGCTCTTTCAGTGATATCCTAAGCACAACGTAACACAGGTGAAATGGGCTGGAGGCACAGTTGTGG TGGAACACGCGGCCAGCAGGACACTGGGACTGATCCCCAGCAGCACAAAGAAAGTGATAGGAACACAGAGC GAGAGTTAGAAGGGACAGGGTCACCGTCAGAGATACGGTGTCTAACTCCTGCAACCCTACCTGTAATTATT CCATATTATAAACATATACTATATAACTGTGGGTCTCTGCATGTTCTAGAATATGAATTCTATTTGATTGT AAAACAAAACTATAAAAATAAGTAAAAAAATAAAAAATAAACAGATACTTAAAATCAAAAAAAAAAAAAAA AAAAAAAAAA Reverse Complement of SEQ ID NO: 1 SEQ ID NO: 5 AATAAGGTATAGTGATACCTCATGTTAAAGTCAATGTGACTTAGTAGTCATCTCCATTTGTTTAAAGACAG CGAACTTATTTTAATACAGTATTTAATTCTGCTTCATTAGGTTTCATAAATAAATCATATCAAACATTTAC TCCAAATTATTTTGTAGCAAAATGAAGCATCAGTTTAAAAATTTGTATTACAGATTTTTACACATACTCTG TGCTGACGATTTTTAGTTTTGAAATTAATACTACAACATTTAAGAACTGTACAATTACCAGTCCTCTGTAT TTAGTCAAGTTTAGAGTTTTAACAAGAGTACTAGTTTTTTAAACTGGGAAAGTTGTGATATTAGCTCATAT GATGCCTTTTAAAATAAAAGTCTCTAAATTTTTTATTTAGAAAAGTTATTTAATAAGTTCACCTATTGATT ATAATCTAGATTGTGACCATCTAAAATTGATTCCCACATCACAAAATTTAAAACAAATAGTATTATAAGAA TTTTGATTGAGAAATGTAAACGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATAGACAGTG ACTTTAAGATAAAAAAAATCTATTTCTCAAGCTTTCTCTTAAGGATTTAATACCAGATTATTAGACCACAT TAACTTGGAATGAGGTTAATGTTTAAATTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTCTGA ATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAAGATA ATCCTCTTCTCCTCTCTGGCTTAGATTTTGCTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCTCCA CACTCATCATGCCACCACCAGCCTCCTGAATAACCCTCTGGACAGTTGAAGTGTCCTTTTGCTTTGTGATC CCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCGCAACTAGAT GTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTCCAG TCTTCCAACTCAATTCGTAAAACATAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAACCA AAATTCTCCATCAAGCCTCCCAAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATCCAT CTATTCGATGTTGAATTAATGTCCATGGACTACCTGATATAACATCACAGTAGACATGAAAAACTTGAGAG TTGCTGGGTCTGATGGCATACATGCCACTTGTATGTTCACCTCTGTTATAAATGGTGGTACATTCAGCAGG AATGCCATCATGTTTTACATTTCTTATTTCATTCAACTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCT TGGAAGATAGAGAAATTTCTGTGGGTTCTTGAATACTAGTCCTTCTGAGCTGATTTTCTATTTCTTTTATT TGACTATGCTGTTGGTTTAATTGTTTATATTGGTCTTCCACGGTCTGGAGAAGGTCTTTGATGCTATTATC TTGTTTTTCTACAAAAGTTTTAAGTGAAGTTACTTCTGGGTGTTCTGGAGTTTCAGGTTGATTTTGAATTA AGTTAGTTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCA AGTTTTGAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCT TCTCAGTTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGCGATAGATCATAAAAAGACTGATCAAATA TGTTGAGTTTTTGAAATATGTCATTAATTTGGCCCTTCGTCTTATGGACAAAGTCTTTAAGACCATGTCCC AACTGAAGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGA TAGAGAATCAAATGATGAATTGTCTTGATCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGA GCTTAATTGTGAACATTTTTATCTTGATTTTCAATTTCAAGCAACGTGGAACTGTTTTCTTCTGGAA Reverse Complement of SEQ ID NO: 2 SEQ ID NO: 6 TAAGGTATAGTGATACCTCATATTAAAGTCAATGTGACTTAGTAATCATCATCTCCATTTGTTTAAAAGAC AGCGAACCTATTTTAATACAGTATTTAATCCTGCTTCATTAGGTTTCATAAATAAATCCTATCAAAAATTT ACTCCAGATTATTTTGTAGCAAAATGAGGCATCAGTTTAAAAATTTATATTACAGATTTTTACACATACTC TGTGCTGACAATTTTTAGTTTTGAAATTAATATTACAACAAATTTAAGAACTGTATAATTATCAGTCCTCT TTATTTAGTCAAGTTTAGAGTTTTAACAAGAAAACTAGTTTTTTAAACTGGGAAAATTGTGATATTAGCTC ATATGATGACTTTTAAAATTAAAGTCTCTAAGTTTTTTATTTAGAAAAGTTATTTAGTAAGTTCACCTGTT GATTATAATTTAGATTGCGACCATCTAAAATTGATTCCCACATCACAAAATATAAAACAAATAGTGTTGTA GGAATTTTGATTGAGAAATGTAAATGGTATTCTTTAAGGTTATGTGATTGTATGTTTAATCTTAAATTGAC AGTGACTTTAAGATAAAAAAATCTATTTCTCAAGCCTTCTCTTAAGGATTTAATACCAGATTATTAAACCA CATTAACTTGGAATGAGTTTAATGTTTAATTTATTGCCTTTTAAATTTGCCTCAGTTCATTCAAAGCTTTC TGAATCTGTTGGATGGATCAACATTTTGGTTGATTTTATAGAGTATAACCTTCCATTTTGAGACTTCCAGG ATAATCCTCTTCTCCGCTCTGGCTTAGATTTTGTTCTTGGTTTGTTATATTTACCATTTAGGTTGTTTTCT CCACACTCATCATGCCACCACCAGCCTCCTGAATAACTCTCTGGACAGCTGAAGTGTCCTTTTGCTTTGTG ATCCCAAGTAGAAAACACCAAATCTTTGTTTTCCGGGATTGCATTGGGGACATTGCCAGTAATCTTAACTA CATGTAGCGTATAGTTGGTTTCGTGATTTCCCAAGTAAAAAGAATATTCAATATAATGTTTGTTGTCTTTC CAGTCTTCCAACTCAATTCGTAAAACGTAATTAGATTGCTTCACTATGGAGTATATCTTCTCTAGGCCCAA CCAGAATTCTCCATCAAGCCTCCCGAAACCATATTTGTAGTTCTCCCACGTTTCATTGAAGTTTTGTGATC CATCTATTCTCCTTAGACAGGTTTTACCTGATACAACATCACAGTAGACATGAAAAACTTGAGAGTTGCTG GGTCTGATGGCATACATGCCACTTATATGTTCACCTCTATTGTAAATGGTGGTACAATCAGCAGGAATGCC ATCATGTTTTACATTTCTTATTTCATTCAGCTGAAGAAAGGGAGTAGTTCTTGGTGCTCTTGGCTTGGAAG ATAGAGAAATTTCTGTGGGTTCTTGAATATTAGTCATTCTGAGCTGATTTTCTATTTCTTTTATTTGACTG TGCTGTTGGTTTAATTGCTTATATTGTTCTTCCACAGTCTGGAGAAGGTCTTTGATGCTATTATCTTGTTT TTCTACAAAACTTTTAAGTGAAGTTACTTCTGGATGTTCTGGAGTTTCAGGTTGATTTTGAATTAAGTTAG TTAGTTGCTCTTCTAAATATTTCACTTTTTGTTGAAGTAGAATTTTTTCTTCTAGGAGGCTTTCAAGTTTT GAGTTGAGTTCAAGTGACATATTCTTTACCTCTTCATTTTTGACTTGTAGTTTATATGTAGTTCTTCTCAG TTCCTTTTCTTCTTCTTTGATTTCACTGGTTTGCAGTGATAGATCATAAAAAGACTGATCAAATATGTTGA GTTTTTGAAATATGTCATTAATTTGGCCCTTAGTCTTATGGACAAAGTCTTTAAGACCATGTCCCAACTGA AGGAGGCCATTGGCTAAAATTTTTACATCGTCTAACATAGCAAATCTTGATTTTGGCTCTGGAGATACAGA ATCAAATGATGAATTGTCTTGGTCAATTCTGGAGGAAATAACTAGAGGAACAATAAAAAGAAGGAGCTTAA TTGTGAACATTTTTATCCTGATTTTCAATTTCAAGCAACGTGGAACTGTGTTCTTCTGGAAGCAGACCTAG ACTTCTTAACTCTATATAT Reverse Complement of SEQ ID NO: 3 SEQ ID NO: 7 CAGGAGGGAGAAGTTCCAAATTGCTTAAAATTGAATAATTGAGACAAAAAATGCACACAATTAAATTATTC CTTTTTGTTGTTCCTTTAGTAATTGCATCCAGAGTGGATCCAGACCTTTCATCATTTGATTCTGCACCTTC AGAGCCAAAATCAAGATTTGCTATGTTGGATGATGTCAAAATTTTAGCGAATGGCCTCCTGCAGCTGGGTC ATGGACTTAAAGATTTTGTCCATAAGACTAAGGGACAAATTAACGACATATTTCAGAAGCTCAACATATTT GATCAGTCTTTTTATGACCTATCACTTCGAACCAATGAAATCAAAGAAGAGGAAAAGGAGCTAAGAAGAAC TACATCTACACTACAAGTTAAAAACGAGGAGGTGAAGAACATGTCAGTAGAACTGAACTCAAAGCTTGAGA GTCTGCTGGAAGAGAAGACAGCCCTTCAACACAAGGTCAGGGCTTTGGAGGAGCAGCTAACCAACTTAATT CTAAGCCCAGCTGGGGCTCAGGAGCACCCAGAAGTAACATCACTCAAAAGTTTTGTAGAACAGCAAGACAA CAGCATAAGAGAACTCCTCCAGAGTGTGGAAGAACAGTATAAACAATTAAGTCAACAGCACATGCAGATAA AAGAAATAGAAAAGCAGCTCAGAAAGACTGGTATTCAAGAACCCTCAGAAAATTCTCTTTCTTCTAAATCA AGAGCACCAAGAACTACTCCCCCTCTTCAACTGAACGAAACAGAAAATACAGAACAAGATGACCTTCCTGC CGACTGCTCTGCCGTTTATAACAGAGGCGAACATACAAGTGGCGTGTACACTATTAAACCAAGAAACTCCC AAGGGTTTAATGTCTACTGTGATACCCAATCAGGCAGTCCATGGACATTAATTCAACACCGGAAAGATGGC TCACAGGACTTCAACGAAACATGGGAAAACTACGAAAAGGGCTTTGGGAGGCTCGATGGAGAATTTTGGTT GGGCCTAGAGAAGATCTATGCTATAGTCCAACAGTCTAACTACATTTTACGACTCGAGCTACAAGACTGGA AAGACAGCAAGCACTACGTTGAATACTCCTTTCACCTGGGCAGTCACGAAACCAACTACACGCTACATGTG GCTGAGATTGCTGGCAATATCCCTGGGGCCCTCCCAGAGCACACAGACCTGATGTTTTCTACATGGAATCA CAGAGCAAAGGGACAGCTCTACTGTCCAGAAAGTTACTCAGGTGGCTGGTGGTGGAATGACATATGTGGAG AAAACAACCTAAATGGAAAATACAACAAACCCAGAACCAAATCCAGACCAGAGAGAAGAAGAGGGATCTAC TGGAGACCTCAGAGCAGAAAGCTCTATGCTATCAAATCATCCAAAATGATGCTCCAGCCCACCACCTAAGA AGCTTCAACTGAACTGAGACAAAATAAAAGATCAATAAATTAAATATTAAAGTCCTCCCGATCACTGTAGT AATCTGGTATTAAAATTTTAATGGAAAGCTTGAGAATTGAATTTCAATTAGGTTTAAACTCATTGTTAAGA TCAGATATCACCGAATCAACGTAAACAAAATTTATC Reverse Complement of SEQ ID NO: 4 SEQ ID NO: 8 TTTTTTTTTTTTTTTTTTTTTTTTTGATTTTAAGTATCTGTTTATTTTTTATTTTTTTACTTATTTTTATA GTTTTGTTTTACAATCAAATAGAATTCATATTCTAGAACATGCAGAGACCCACAGTTATATAGTATATGTT TATAATATGGAATAATTACAGGTAGGGTTGCAGGAGTTAGACACCGTATCTCTGACGGTGACCCTGTCCCT TCTAACTCTCGCTCTGTGTTCCTATCACTTTCTTTGTGCTGCTGGGGATCAGTCCCAGTGTCCTGCTGGCC GCGTGTTCCACCACAACTGTGCCTCCAGCCCATTTCACCTGTGTTACGTTGTGCTTAGGATATCACTGAAA GAGCTCTATACTGCTATCTTTGCAGCTCATTTGTAGCATGGCCCCTCACTAGAGCCAATGGCGTATACTAT GTTCCTTAATAAGACTTATGATAACCATGAACTATAGAATTAAGTTTCAACAAGCATACTGCAGGCATGCA GTAGTGATAAGTACAATGAGATCATTGCAGTTGCTATCATGGATAATATTTACCAATCAGTTTTATGAAGA CTGGGGTCTGCTTTTGCTTATTGTTACATTTCCACAGCCCATTTTATAATTGCTGCTTAATTTATAATTCT TAATGTCGAGAAGGAGTATCAATGTGTGGCCCAGACTGGTCTTGAACTTTCAATTCACCTGCTGCTCCTCC TCCCCCCTTGCCCAAAAGCGCTATGGTCTCAGGCCTGTGTCAACACACCCAACTAATACTATTAAATCAAC AGATAAGTGAGTGATGCAAGGAAATCACTTTACCATCAAGCCTCCCAAAACCCTTTTCGTAGTTTTCCCAC GTTTGGTTGAAGTTTTGAGAGCCATCTTTCCGGTGTTGAATTAATGTCCGTGGAGTGCCTGATTGGGTGTC ACAGTAGACATTAAACACTTGAGAGCTGCTTGGTCTAATAGTATACACGCCACTTGTATGTTCACCTCTGT TATAAATGGCAGAGCAGTCAGCAGGCAGATCATCTTGTTCTATATTTTTTGCTTCCTTCAGATGAAGAGGG GGAGTAGTTCTTGGTGCTCTTGGTTTAGAATAAAGAGAATTTTCAGTGGGTTCTTGAATGCCAGTCTTTCT GAGCTGATTTTCTATTTCTTTTATCTGAATGTGCTGTTGACTTAGTTGTTTATATTGTTCTTCCACACTCT GGAGGAGTTCTCTTATGCTGTTATCTTGCTGTTCTACAAAACTTTTAAGTGACGTTACCTCTGGGTGCTCC CGAGCCCCAGGCGGGTTCTGAACCAAGCTGGTCAGCTGTTCCTCCAAAGCCCTGACTCTGTGTTGGAGCGC CATCTTCTCCTCCAGTAGACTTTCAAGCTTTGAGTTCAGTTCAAGTGACATATTCTTCACCTCTTCGTTTT TAACTTGTAGTTTAGATGTGGTTCTTCTTAGCTCCTTTTCCTCTTCTTTGATTTCATTGGTTTGAAGTGAT AGGTCATAAAAACACTGATCAAATATGTTGAGCTTCTGAAATATGTCATTAATTTGTCCCTTTGTCTTATG GACAAAATCTTTAAGACCATGACCCAGCTGCAGGAGGCCATTGGCTAAAATTTTGACATCATCCAACATAG CAAATCTTGATTTTGGCTCTGACGGTACAGAATCAAATGGCGAAAGGTCTGGATCAACTCTGGACGAAATT ACTAGAGGAACAACAAAAAGGAGCAGCTTAATTGTGTGCATTTTTGTTTCAATTATTCAATTTCAAGCAAT TTGGAACGTC Macaca fascicularis angiopoietin-like 3 (Angptl3), mRNA SEQ ID NO: 9 GGGTAGTATATAGAGTTAAGAAGTCTAGGTCTGCTTCCAGAAGAACACAGTTCCACGCTGCTTGAAATTGA AAATCAGGATAAAAATGTTCACAATTAAGCTCCTTCTTTTTATTGTTCCTCTAGTTATTTCCTCCAGAATT GACCAAGACAATTCATCATTTGATTCTGTATCTCCAGAGCCAAAATCAAGATTTGCTATGTTAGACGATGT AAAAATTTTAGCCAATGGCCTCCTTCAGTTGGGACATGGTCTTAAAGACTTTGTCCATAAGACTAAGGGCC AAATTAATGACATATTTCAAAAACTCAACATATTTGATCAGTCTTTTTATGATCTATCACTGCAAACCAGT GAAATCAAAGAAGAAGAAAAGGAACTGAGAAGAACTACATATAAACTACAAGTCAAAAATGAAGAGGTAAA GAATATGTCACTTGAACTCAACTCAAAACTTGAAAGCCTCCTAGAAGAAAAAATTCTACTTCAACAAAAAG TGAAATATTTAGAAGAGCAACTAACTAACTTAATTCAAAATCAACCTGCAACTCCAGAACATCCAGAAGTA ACTTCACTTAAAAGTTTTGTAGAAAAACAAGATAATAGCATCAAAGACCTTCTCCAGACTGTGGAAGAACA ATATAAGCAATTAAACCAACAGCATAGTCAAATAAAAGAAATAGAAAATCAGCTCAGAATGACTAATATTC AAGAACCCACAGAAATTTCTCTATCTTCCAAGCCAAGAGCACCAAGAACTACTCCCTTTCTTCAGCTGAAT GAAATAAGAAATGTAAAACATGATGGCATTCCTGCTGATTGTACCACCATTTACAATAGAGGTGAACATAT AAGTGGCACGTATGCCATCAGACCCAGCAACTCTCAAGTTTTTCATGTCTACTGTGATGTTGTATCAGGTA GTCCATGGACATTAATTCAACATCGAATAGATGGATCACAAAACTTCAATGAAACGTGGGAGAACTACAAA TATGGTTTCGGGAGGCTTGATGGAGAATTCTGGTTGGGCCTAGAGAAGATATACTCCATAGTGAAGCAATC TAATTACGTTTTACGAATTGAGTTGGAAGACTGGAAAGACAACAAACATTATATTGAATATTCTTTTTACT TGGGAAATCACGAAACCAACTATACGCTACATGTAGTTAAGATTACTGGCAATGTCCCCAATGCAATCCCG GAAAACAAAGATTTGGTGTTTTCTACTTGGGATCACAAAGCAAAAGGACACTTCAGCTGTCCAGAGAGTTA TTCAGGAGGCTGGTGGTGGCATGATGAGTGTGGAGAAAACAACCTAAATGGTAAATATAACAAACCAAGAA CAAAATCTAAGCCAGAGCGGAGAAGAGGATTATCCTGGAAGTCTCAAAATGGAAGGTTATACTCTATAAAA TCAACCAAAATGTTGATCCATCCAACAGATTCAGAAAGCTTTGAATGAACTGAGGCAAATTTAAAAGGCAA TAAATTAAACATTAAACTCATTCCAAGTTAATGTGGTTTAATAATCTGGTATTAAATCCTTAAGAGAAGGC TTGAGAAATAGATTTTTTTATCTTAAAGTCACTGTCAATTTAAGATTAAACATACAATCACATAACCTTAA AGAATACCATTTACATTTCTCAATCAAAATTCTTACAACACTATTTGTTTTATATTTTGTGATGTGGGAAT CAATTTTAGATGGTCGCAATCTAAATTATAATCAACAGGTGAACTTACTAAATAACTTTTCTAAATAAAAA ACTTAGAGACTTTAATTTTAAAAGTCATCATATGAGCTAATGTCACAATTTTCCCAGTTTAAAAAACTAGT TTTCTTGTTAAAACTCTAAACTTGACTAAATAAAGAGGACTGATAATTATACAGTTCTTAAATTTGTTGTA ATATTAATTTCAAAACTAAAAATTGTCAGCACAGAGTATGTGTAAAAATCTGTAATATAAATTTTTAAACT GATGCCTCATTTTGCTACAAAATAATCTGGAGTAAATTTTTGATAGGATTTATTTATGAAACCTAATGAAG CAGGATTAAATACTGTATTAAAATAGGTTCGCTGTCTTTTAAACAAATGGAGATGATGATTACTAAGTCAC ATTGACTTTAATATGAGGTATCACTATACCTTAACATATTTGTTAAAACGTATACTGTATACATTTTGTGT 

We claim:
 1. A double-stranded ribonucleic acid (dsRNA) agent for inhibiting expression of Angiopoietin-like 3 (ANGPTL3), wherein said dsRNA agent comprises a sense strand and an antisense strand forming a double stranded region, the antisense strand comprising a region of complementarity to an mRNA encoding ANGPTL3 which comprises at least 15 contiguous nucleotides differing by no more than 3 nucleotides from the complement of nucleotides 1051-1098 of SEQ ID NO:1.
 2. The dsRNA agent of claim 1, wherein said dsRNA agent comprises at least one modified nucleotide.
 3. The dsRNA agent of claim 2, wherein at least one of said modified nucleotides is selected from the group consisting of a 2′-O-methyl modified nucleotide, a nucleotide comprising a 5′-phosphorothioate group, and a terminal nucleotide linked to a cholesteryl derivative or a dodecanoic acid bisdecylamide group.
 4. The dsRNA agent of claim 1, wherein the region of complementarity is at least 17 nucleotides in length.
 5. The dsRNA agent of claim 1, wherein each strand is no more than 30 nucleotides in length.
 6. The dsRNA agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 1 nucleotide.
 7. The dsRNA agent of claim 1, further comprising a ligand.
 8. A cell containing the dsRNA agent of claim
 1. 9. A pharmaceutical composition for inhibiting expression of an ANGPTL3 gene comprising the dsRNA agent of claim
 1. 10. A method of inhibiting ANGPTL3 expression in a cell, the method comprising: (a) contacting the cell with the dsRNA agent of claim 1; and (b) maintaining the cell produced in step (a) for a time sufficient to obtain degradation of the mRNA transcript of an ANGPTL3gene, thereby inhibiting expression of the ANGPTL3 gene in the cell.
 11. The method of claim 10, wherein said cell is within a subject.
 12. A method of treating a subject having a disorder that would benefit from reduction in ANGPTL3 expression, comprising administering to the subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby treating said subject.
 13. The method of claim 12, wherein the disorder is a disorder of lipid metabolism.
 14. A method of inhibiting the expression of ANGPTL3 in a subject, the method comprising administering to said subject a therapeutically effective amount of the dsRNA agent of claim 1, thereby inhibiting the expression of ANGPTL3 in said subject.
 15. The dsRNA agent of claim 1, wherein the double-stranded region is 17-25 nucleotide pairs in length.
 16. The dsRNA agent of claim 1, wherein the double-stranded region is 21-23 nucleotide pairs in length.
 17. The dsRNA agent of claim 1, wherein each strand is 19-30 nucleotides in length.
 18. The dsRNA agent of claim 1, wherein the sense strand has a total of 21 nucleotides and the antisense strand has a total of 23 nucleotides.
 19. The dsRNA agent of claim 1, wherein the region of complementarity is between 19 and 21 nucleotides in length.
 20. The dsRNA agent of claim 3, wherein said modified nucleotide is a 2′-O-methyl or a 2′-fluoro modified nucleotide.
 21. The dsRNA agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides.
 22. The dsRNA agent of claim 7, wherein the ligand is conjugated to the 3′ end of the sense strand of the dsRNA agent.
 23. The dsRNA agent of claim 22, wherein the ligand is an N-acetylgalactosamine (GalNAc) derivative.
 24. The dsRNA agent of claim 22, wherein the ligand is


25. The dsRNA agent of claim 1, wherein said dsRNA agent further comprises at least one phosphorothioate or methylphosphonate internucleotide linkage.
 26. The dsRNA agent of claim 25, wherein the phosphorothioate or methylphosphonate internucleotide linkage is at the 3′-terminus of one strand.
 27. The dsRNA agent of claim 1, wherein the region of complementarity comprises any one of the antisense nucleotide sequences selected from the group consisting of 5′-UUGUCUUUCCAGUCUUCCAACUC-3′ (SEQ ID NO:562); 5′-AAUAUAAUGUUUGUUGUCUUUCC-3′ (SEQ ID NO:449); 5′-UUCAAUAUAAUGUUUGUUGUCUU-3′ (SEQ ID NO:498); 5′-AUUCAAUAUAAUGUUUGUUGUCU-3′ (SEQ ID NO:480); 5′-UAUUCAAUAUAAUGUUUGUUGUC-3′ (SEQ ID NO:575); 5′-AUAUUCAAUAUAAUGUUUGUUGU-3′ (SEQ ID NO:486); 5′-AAUAUUCAAUAUAAUGUUUGUUG-3′ (SEQ ID NO:495); 5′-AAGAAUAUUCAAUAUAAUGUUUG-3′ (SEQ ID NO:582); 5′-AAAGAAUAUUCAAUAUAAUGUUU-3′ (SEQ ID NO:533); and 5′-AAAAGAAUAUUCAAUAUAAUGUU-3′ (SEQ ID NO:459).
 28. The dsRNA agent of claim 1, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of 5′-GUUGGAAGACUGGAAAGACAA-3′ (SEQ ID NO:377) and 5′-UUGUCUUUCCAGUCUUCCAACUC-3′ (SEQ ID NO:562); 5′-AAAGACAACAAACAUUAUAUU-3′ (SEQ ID NO:264) and 5′-AAUAUAAUGUUUGUUGUCUUUCC-3′ (SEQ ID NO:449); 5′-GACAACAAACAUUAUAUUGAA-3′ (SEQ ID NO:313) and 5′-UUCAAUAUAAUGUUUGUUGUCUU-3′ (SEQ ID NO:498); 5′-ACAACAAACAUUAUAUUGAAU-3′ (SEQ ID NO:295) and 5′-AUUCAAUAUAAUGUUUGUUGUCU-3′ (SEQ ID NO:480); 5′-CAACAAACAUUAUAUUGAAUA-3′ (SEQ ID NO:390) and 5′-UAUUCAAUAUAAUGUUUGUUGUC-3′ (SEQ ID NO:575); 5′-AACAAACAUUAUAUUGAAUA-3′ (SEQ ID NO:301) and 5′-AUAUUCAAUAUAAUGUUUGUUGU-3′ (SEQ ID NO:486); 5′-ACAAACAUUAUAUUGAAUAUU-3′ (SEQ ID NO:310) and 5′-AAUAUUCAAUAUAAUGUUUGUUG-3′ (SEQ ID NO:495); 5′-AACAUUAUAUUGAAUAUUCUU-3′ (SEQ ID NO:397) and 5′-AAGAAUAUUCAAUAUAAUGUUUG-3′ (SEQ ID NO:582); 5′-ACAUUAUAUUGAAUAUUCUUU-3′ (SEQ ID NO:348) and 5′-AAAGAAUAUUCAAUAUAAUGUUU-3′ (SEQ ID NO:533); and 5′-CAUUAUAUUGAAUAUUCUUUU-3′ (SEQ ID NO:274) and 5′-AAAAGAAUAUUCAAUAUAAUGUU-3′ (SEQ ID NO:459).
 29. The dsRNA agent of claim 28, wherein the sense and antisense strands comprise nucleotide sequences selected from the group consisting of 5′-GfuUfgGfaAfgAfCfUfgGfaAfaGfaCfaAf (SEQ ID NO: 747) and 5′-uUfgUfcUfuUfcCfaguCfuUfcCfaAfcsUfsc (SEQ ID NO:932); 5′-AfaAfgAfcAfaCfAfAfaCfaUfuAfuAfuUf (SEQ ID NO:634) and 5′-aAfuAfuAfaUfgUfuugUfuGfuCfuUfusCfsc (SEQ ID NO:819); 5′-GfaCfaAfcAfaAfCfAfuUfaUfaUfuGfaAf (SEQ ID NO:683) and 5′-uUfcAfaUfaUfaAfuguUfuGfuUfgUfcsUfsu (SEQ ID NO:868); 5′-AfcAfaCfaAfaCfAfUfuAfuAfuUfgAfaUf (SEQ ID NO:665) and 5′-aUfuCfaAfuAfuAfaugUfuUfgUfuGfusCfsu (SEQ ID NO:850); 5′-CfaAfcAfaAfcAfUfUfaUfaUfuGfaAfuAf (SEQ ID NO:760) and 5′-uAfuUfcAfaUfaUfaauGfuUfuGfuUfgsUfsc (SEQ ID NO:945); 5′-AfaCfaAfaCfaUfUfAfuAfuUfgAfaUfaUf (SEQ ID NO:671) and 5′-aUfaUfuCfaAfuAfuaaUfgUfuUfgUfusGfsu (SEQ ID NO:856); 5′-AfcAfaAfcAfuUfAfUfaUfuGfaAfuAfuUf (SEQ ID NO:680) and 5′-aAfuAfuUfcAfaUfauaAfuGfuUfuGfusUfsg (SEQ ID NO:865); 5′-AfaCfaUfuAfuAfUfUfgAfaUfaUfuCfuUf (SEQ ID NO:767) and 5′-aAfgAfaUfaUfuCfaauAfuAfaUfgUfusUfsg (SEQ ID NO:952); 5′-AfcAfuUfaUfaUfUfGfaAfuAfuUfcUfuUf (SEQ ID NO:718) and 5′-aAfaGfaAfuAfuUfcaaUfaUfaAfuGfusUfsu (SEQ ID NO:903); and 5′-CfaUfuAfuAfuUfGfAfaUfaUfuCfuUfuUf (SEQ ID NO:644) and 5′-aAfaAfgAfaUfaUfucaAfuAfuAfaUfgsUfsu (SEQ ID NO:829), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; Af, Gf, Cf and Uf are 2′-fluoro A, G, C and U; and s is a phosphorothioate linkage.
 30. The pharmaceutical composition of claim 9, further comprising a lipid formulation.
 31. The pharmaceutical composition of claim 9, wherein the dsRNA agent is present in a buffered solution. 